Methods of prevention and treatment of inflammatory bowel disease

ABSTRACT

The present invention is based upon methods of treating inflammatory conditions in the intestinal tract of mammals using growth factor related polypeptides. The invention includes methods of reducing the morality rate or delaying mortality in a subject suffering from an inflammatory pathology. Methods of using fibroblast growth factor-CX (FGF-CX) polynucleotide sequences and the FGF-CX polypeptides encoded by such nucleic acid sequences, or variants, fragments and homologs thereof, are claimed in the invention. Similarly, methods of using FCTRX polynucleotide sequences and the FCTRX polypeptides encoded by such nucleic acid sequences, or variants, fragments and homologs thereof, alone or in combination, are also claimed in the invention. FCTRX collectively refers to any of six variant FCTRX sequences, variously designated FCTR1, FCTR2, FCTR3, FCTR4, FCTR5 and FCTR6.

RELATED APPLICATION

This application claims the benefit of priority from U.S. Provisionalapplication Ser. No. 60/246,206 filed Nov. 6, 2000, and is acontinuation-in-part of U.S. Non-Provisional application Ser. No.09/992,840, filed Nov. 6, 2001, the contents of which are incorporatedherein in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to methods of treatment ofinflammatory conditions in the intestinal tract of mammals using growthfactor related polypeptides. More specifically, the polypeptidesemployed in the methods of the invention are related to a member of thefibroblast growth factor family and to a member of the platelet derivedgrowth factor family.

BACKGROUND OF THE INVENTION

Inflammatory bowel disease comprises two distinct subsets: ulcerativecolitis and Crohn's disease. In 1999, approximately 1.7 million peoplewere diagnosed with this debilitating disease. Satisfactory treatment ofIBD is an unmet medical need, as existing therapeutics have not beensuccessful in curtailing the disease and preventing surgeries. Up toforty percent of all ulcerative colitis patients undergo surgery, whichtypically includes the removal of part of the large intestine or a fullcolostomy. Such surgery is not curative for Crohn's disease, as 75% ofall patients undergo at least one surgery in their lifetime, and up to90% of these patients require additional surgeries. Consequently atherapeutic that can successfully treat inflammatory bowel disease willhave the beneficial effects of improving a patient's quality of life,while potentially saving the healthcare system millions of dollars incosts associated with invasive surgical procedures.

SUMMARY OF THE INVENTION

The present invention is based upon methods of treating inflammatoryconditions in the intestinal tract of mammals using growth factorrelated polypeptides. Methods of using fibroblast growth factor-CX(FGF-CX) polynucleotide sequences and the FGF-CX polypeptides encoded bysuch nucleic acid sequences, or variants, fragments and homologsthereof, are claimed in the invention. Similarly, methods of using FCTRXpolynucleotide sequences and the FCTRX polypeptides encoded by suchnucleic acid sequences, or variants, fragments and homologs thereof,alone or in combination, are also claimed in the invention. FCTRXcollectively refers to any of six variant FCTRX sequences, designatedFCTR1, FCTR2, FCTR3, FCTR4, FCTR5 and FCTR6.

In one aspect, the invention provides a method of promoting the growthof a population of cells whereby the cells are placed into contact witha composition including a FGF-CX or FCTRX polypeptide, or a compositionincluding FGF-CX and FCTRX polypeptides. In another aspect, theinvention provides a method of treating an inflammatory pathology in asubject, whereby an FGF-CX or an FCTRX polypeptide composition isadministered to the subject. In yet another aspect, the inventionprovides a method of delaying the onset of an inflammatory pathology ina subject, whereby a composition including a FGF-CX or FCTRXpolypeptide, or a composition including FGF-CX and FCTRX polypeptides,is administered to the subject. In a further aspect, the inventionprovides a method of ameliorating an inflammatory pathology in asubject, whereby a composition including a FGF-CX or FCTRX polypeptide,or a composition including FGF-CX and FCTRX polypeptides, isadministered to the subject.

In one embodiment, the subject is a mammal. In another embodiment, thesubject is human. In yet another embodiment, the inflammatory pathologyis inflammatory bowel disease, an inflammatory condition occurring inthe colon, an inflammatory condition occurring in the small intestine,or Crohn's disease. In yet another embodiment, the FGF-CX polypeptide isgiven by SEQ ID NO:2, or a variant, deletion mutant, or a variant of thedeletion mutant thereof, wherein up to 15% of the residues of eithervariant are changed according to a conservative amino acid substitution.In still yet another embodiment, the FCTRX polypeptide is given by anyone of SEQ ID NOS:4, 6, 8, 10, 12, and 14, or a variant, deletionmutant, variant of the deletion mutant, p35 form, or a variant of thep35 form thereof, wherein up to 15% of the residues of any variant arechanged according to a conservative amino acid substitution. In yet afurther embodiment, the polypeptide composition is administeredintravenously or subcutaneously.

The invention further provides a method of preparing a pharmaceuticalcomposition, whereby a polypeptide effective in treating an inflammatorypathology is combined with a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition is suitable forintravenous, or subcutaneous administration to the subject. In anotherembodiment, the polypeptide is FGF-CX. In yet another embodiment, theFGF-CX polypeptide is given by SEQ ID NO:2, or a variant, deletionmutant, or a variant of the deletion mutant thereof, wherein up to 15%of the residues of either variant are changed according to aconservative amino acid substitution. In a further embodiment, thepolypeptide is FCTRX. In yet a further embodiment, the FCTRX polypeptideis given by any one of SEQ ID NOS: 4, 6, 8, 10, 12, and 14, or avariant, deletion mutant, variant of the deletion mutant, p35 form, or avariant of the p35 form thereof, wherein up to 15% of the residues ofany variant are changed according to a conservative amino acidsubstitution. In yet a further embodiment, the inflammatory pathology isinflammatory bowel disease, an inflammatory condition occurring in thecolon, an inflammatory condition occurring in the small intestine, orCrohn's disease.

Contemplated within the invention is a method of reducing the mortalityrate in a subject suffering from an inflammatory pathology comprisingadministering to the subject a composition comprising a firstpolypeptide wherein the first polypeptide comprises either a FGFCXpolypeptide or a FCTRX polypeptide. A method of delaying mortality in asubject suffering from an inflammatory pathology comprisingadministering to the subject a composition comprising a firstpolypeptide wherein the first polypeptide comprises either a FGFCXpolypeptide or a FCTRX polypeptide.

In one embodiment, a method of reducing the mortality rate or delayingmortality in a subject suffering from an inflammatory pathology includesproviding a FGF-CX polypeptide or a FCTRX polypeptide suitable forintravenous, or subcutaneous administration to the subject. In anotherembodiment, the subject is mammalian. In a more specific embodiment, thesubject is human. In a different embodiment, the polypeptide is FGF-CX.In further embodiments, the FGF-CX polypeptide includes at least one ofthe polypeptide of SEQ ID NO:2, or a variant, deletion mutant, or avariant of the deletion mutant thereof, wherein up to 15% of theresidues of either variant are changed according to a conservative aminoacid substitution. In another embodiment, the polypeptide is FCTRX. Inyet further embodiments, the FCTRX polypeptide is given by at least oneof SEQ ID NOS: 4, 6, 8, 10, 12, and 14, or a variant, deletion mutant,variant of the deletion mutant, p35 form, or a variant of the p35 formthereof, wherein up to 15% of the residues of any variant are changedaccording to a conservative amino acid substitution. In yet a furtherembodiment, the inflammatory pathology is at least one of aninflammatory bowel disease, an inflammatory condition occurring in thecolon, an inflammatory condition occurring in the small intestine, orCrohn's disease.

Contemplated disorders within the invention include pathology such asinflammatory conditions in the gastrointestinal tract, including but notlimited to inflammatory bowel disease such as ulcerative colitis andCrohn's disease, growth and proliferative diseases such as cancer,angiogenesis, atherosclerotic plaques, collagen formation, cartilage andbone formation, cardiovascular and fibrotic diseases and diabeticulcers. In addition, FCTRX nucleic acids and their encoded polypeptideswill be therapeutically useful for the prevention of aneurysms and theacceleration of wound closure through gene therapy. Furthermore, FCTRXnucleic acids and their encoded polypeptides can be utilized tostimulate cellular growth. wound healing, neovascularization and tissuegrowth, and similar tissue regeneration needs. More specifically, aFCTRX nucleic acid or polypeptide may be useful in treatment of anemiaand leukopenia, intestinal tract sensitivity and baldness. Treatment ofsuch conditions may be indicated, e.g., in patients having undergoneradiation or chemotherapy, wherein treatment would minimize anyhyperproliferative side effects.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Other features and advantages of the invention will beapparent from the following detailed description and claims.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Western analysis of FGF-CX protein secreted by 293 cells.

FIG. 2 shows a Western analysis of FGF-CX protein expressed in E. colicells.

FIG. 3 shows a Western analysis of FGF-CX. Samples from 293 cells (PanelA) or NIH 3T3 cells (Panel B) transiently transfected with the indicatedconstruct were examined by Western analysis using anti-V5 antibody.CM=conditioned media, SE=suramin-extracted conditioned media. Molecularmass markers are indicated on the left.

FIG. 4 presents an image of a Coomassie Blue stained SDS-PAGE gel ofpurified samples of FGF-CX prepared under reducing and nonreducingconditions.

FIG. 5 provides the results of a dose titration growth experimentcarried out using 786-0 human renal carcinoma cells. In this experimentincorporation of bromodeoxyuridine induced by varying amounts of FGF-CX(designated in FIG. 5 as 20858) was determined.

FIG. 6 shows the results of experiments assessing the receptor bindingspecificity of FGF-CX. NIH 3T3 cells were serum-starved, incubated withthe indicated growth factor (square=PDGF-BB; triangle=aFGF;circle=FGF-CX) either alone or together with the indicated soluble FGFR,and analyzed by a BrdU incorporation assay. Experiments were performedin triplicate and are represented as the percent BrdU increase inincorporation of BrdU relative to cells receiving the growth factoralone.

FIG. 7 shows an image of a Coomassie Blue stained SDS-PAGE gel of thearginine supernatant obtained when plasmid pET24a-FGF20X-del54-codon wasexpressed in E.coli strain BL21 (DE3).

FIG. 8 displays the biological activity of a truncated form ofrecombinant FGF-CX (denoted by (d1-23)FGF20 in the FIG.) as representedby its effects on DNA synthesis, compared to that of full length FGF-CX(denoted FGF20 in the FIG.). NIH 3T3 mouse fibroblasts wereserum-starved, incubated with the indicated factor for 18 hr, andanalyzed by a BrdU incorporation assay.

FIG. 9 is a representation of a Western blot of a 30664188.m99 proteinexpressed in E. coli cells.

FIG. 10 is a representation of a Western blot of a 30664188.m99 proteinsecreted by human 293 cells.

FIG. 11. Panel A is a schematic representation of a scheme for therecombinant production, purification and apparent molecular weight of amature form of the protein of clone 30664188.0.99. Panel B includesrepresentations of two Western blot analyses showing expression of a30664188.0.m99 polypeptide.

FIG. 12 is a graph showing incorporation of BrdU into NIH 3T3 cells andCCD-1070 cells in response to various treatments.

FIG. 13 is a graph showing proliferation of NIH 3T3 5-24 cells inresponse to various treatments.

FIG. 14 is a graph showing cell number in NIH 3T3 cells exposed to amock treatment or 30664188.

FIG. 15 is a depiction of a photomicrograph showing cell density andcell morphology of NIH 3T3 cells in response to treatment with pCEP4secCM or 30664188 protein.

FIG. 16 is a depiction of a photomicrograph showing changes in cellnumber in NHost osteoblast cells in response to various treatments.

FIG. 17. Panel A is a representation of a western blot of 30664188.m99expressed by HEK 293 cells cultured in the absence of serum. Panel B isa representation of SDS-PAGE 30664188.m99 protein expressed by HEK 293cells cultured in the presence of serum.

FIG. 18 is a representation of dose titration of BrdU incorporation intoNIH 3T3 cells stimulated by p85 (bars 4-10) and by the p35 fragment of30664188.m99 protein (bars 11-17).

FIG. 19 is a representation of a Western blot and SDS PAGE analysis ofPDGF D. In Panel A, samples from the conditioned medium of HEK 293 cellstransiently transfected with pCEP4/Sec (lane 1) or pCEP4/Sec-PDGF D(lanes 2 & 3) and cultured in the presence (lane 3) or absence (lanes 1& 2) of FBS were examined by SDS-PAGE under reducing conditions,followed by immunoblot analysis using anti-V5 antibody. In Panel B,purified PDGF-D from pCEP4/Sec-PDGF D transfected HEK 293 cells culturedin the presence (lanes 3 & 4) or absence (lanes 1 & 2) of FBS wasresolved by SDS-PAGE and stained with Coomassie Blue. Samples weretreated with (+) and without (−) DTT. Molecular weight markers areindicated on the left.

FIG. 20 is a representation of fragments obtained from p35 andidentified by N-terminal sequencing. In each panel, the upper sequencein black is the predicted sequence from the clone, and the lowersequence in gray is the sequence provided by N-terminal sequencing. Thediagonal shadings represent two fragments of p35. Horizontal shadingrepresents the V5 epitope and vertical shading represents the 6His tag,both of which originate from vector pCEP4/Sec-30664188 (Example 3). InPanel A, two sequences were identified, one beginning with GlyArg (shownwith these two residues underlined), and the second beginning with thethird residue, Ser.

FIG. 21 is a depiction of the SDS-PAGE of the 30664188 gene product inthe presence of fetal bovine serum (Panel B) and Calf Serum (Panel A).Lanes 1 and 2 in each panel show authentic 30664188 p35 alone or in thepresence of serum, respectively. Lane 3 in each panel shows p85 in theabsence of serum, and lanes 4-6 show p85 in the presence of increasingconcentrations of the respective serum.

FIG. 22 is a depiction of the stimulation of the growth of pulmonaryartery smooth muscle cells by growth factors. Smooth muscle cells weretreated with purified p35 PDGF DD, PDGF AA or PDGF BB at theconcentrations indicated, and the amount of BrdU incorporated into DNAwas determined.

FIG. 23 is a diagram showing the proliferation of pulmonary arterysmooth muscle cells in response to various treatments.

FIG. 24 presents bar graphs representing mean body weights of mice onday 0, and on day 6 after various treatments.

FIG. 25 presents bar graphs representing changes in mean body weights ofmice between day 0 and day 6 after various treatments.

FIG. 26 presents bar graphs representing percent changes in mean bodyweights of mice between day 0 and day 6 after various treatments.

FIG. 27 presents bar graphs representing changes in mean weights of thespleens of mice between day 0 and day 6 after various treatments.

FIG. 28 presents bar graphs representing changes in mean spleen weightsof mice between day 0 and day 6 after various treatments.

FIG. 29 presents bar graphs representing changes in mean colon weightsof mice between day 0 and day 6 after various treatments.

FIG. 30 presents bar graphs representing changes in mean colon weightsof mice between day 0 and day 6 after various treatments.

FIG. 31 presents bar graphs representing percent changes in mean colonweights of mice between day 0 and day 6 after various treatments.

FIG. 32 presents bar graphs representing changes in mean colon lengthsof mice between day 0 and day 6 after various treatments.

FIG. 33 presents bar graphs representing percent changes in mean colonlengths of mice between day 0 and day 6 after various treatments.

FIG. 34 presents bar graphs representing mean colon blood content scoresin mice after various treatments.

FIG. 35 presents bar graphs representing mean colon edema scores in miceafter various treatments.

FIG. 36 presents bar graphs representing mean colon inflammation scoresin mice after various treatments.

FIG. 37 presents bar graphs representing mean colon epithelial lossscores in mice after various treatments.

FIG. 38 presents bar graphs representing mean colon erosion contentscores in mice after various treatments.

FIG. 39 presents bar graphs representing sum of histopathology scores inmice after various treatments.

FIG. 40 presents bar graphs representing histopathology scoredifferencess in mice after various treatments.

FIG. 41 presents bar graphs representing mean splenic lymphoid atrophyscores in mice after various treatments.

FIG. 42 presents photomicrographs at 400× in the original image of mousecolon cross sections. Panel A, DSS plus Vehicle; Panel B, DSS+AB020858;Panel C, Normal mouse.

FIG. 43 presents photomicrographs at 50× in the original image of mousecolon crossections. Panel A, DSS plus Vehicle; Panel B, DSS+AB020858;Panel C, Normal mouse.

FIG. 44 presents the change in mean body weight from day 0 upon treatingmice with varying doses of AB020858.

FIG. 45 presents the percent change in mean body weight from day 0 upontreating mice with varying doses of AB020858.

FIG. 46 presents mean colon blood content score upon treating mice withvarying doses of AB020858.

FIG. 47 presents mean colon lengths upon treating mice with varyingdoses of AB020858.

FIG. 48 presents mean colon lengths as a percent of normal, upontreating mice with varying doses of AB020858.

FIG. 49 presents mean colon weights upon treating mice with varyingdoses of AB020858.

FIG. 50 presents mean colon colon weights as a percent of normal, upontreating mice with varying doses of AB020858.

FIG. 51 presents mean spleen weights upon treating mice with varyingdoses of AB020858.

FIG. 52 presents mean distal colon inflammation score upon treating micewith varying doses of AB020858.

FIG. 53 presents mean distal colon gland loss score upon treating micewith varying doses of AB020858.

FIG. 54 presents mean distal colon erosion score upon treating mice withvarying doses of AB020858.

FIG. 55 presents mean sums of histopathology scores upon treating micewith varying doses of AB020858.

FIG. 56 presents mean splenic lymphoid atrophy score upon treating micewith varying doses of AB020858.

FIG. 57 presents mean splenic extramedullary hematopoiesis score upontreating mice with varying doses of AB020858.

FIG. 58 presents the effect of CG53135 Treatment on Weight Loss inIndomethacin-treated rats. Body weight change from Day 0 to Day 5 isshown in grams.

FIG. 59 presents the effect of CG53135 Treatment on Small IntestineWeight in Indomethacin-treated rats.

FIG. 60 presents effect of CG53135 Treatment on absolute neutrophil andlymphocyte counts in indomethacin-treated rats. Blood was collected onDay 5 at necropsy and the cell counts were determined.

FIG. 61 presents effect of CG53135 Treatment on Histopathology Scores inIndomethacin-treated rats. Five sections of affected intestine wereevaluated and scored for necrosis and inflammation as described in themethods.

FIG. 62 presents images showing the protective effect of CG53135 onintestinal architecture. Panel A: Small intestine from normal controlanimal treated iv with vehicle (BSA). Panel. B: Small intestine fromindomethacin-treated rat, further treated with vehicle (BSA) iv. PanelC: Small intestine from indomethacin-treated rat further treated withCG53135, 0.2 mg/kg iv. Sections were stained with H&E and visualized ata magnification of 25). FIG. 62 shows the protective Effect of CG53135on Intestinal Architecturein indomethacin treated rats. Panel A, normalcontrol; Panel B, disease control (indomethacin treated); Panel C,disease model animal treated with 0.2 mg/kg iv CG53135. Photomicrographswere obtained on sections stained with hemotoxylin and eosin, at 25×magnification.

FIG. 63 shows the effect of CG53135 treatment on BrdU Labeling in theIntestine. BrdU incorporation was detected by Immunoperoxidase staining.Panel A: Small intestine from normal control animal (100×). Panel B:Small intestine from indomethacin+vehicle (BSA) treated animal (50×).Panel C: Small intestine from indomethacin+CG53135 0.2 mg/kg iv treatedrat (50×).

FIG. 64. Effect of therapeutically-administered CG53135 on survival inthe DSS model of colitis. Female Balb/c mice were exposed to 4% DSS indrinking water for 7 days (day 0 to day 6) and then switched to normaldrinking water for 4 additional days (day 7 to day 10). CG53135 isidentified as FGF-20 in FIG. 64. Disease control animals (n=9) receiveddaily SC injections of vehicle solution on day 4 to day 9. CG53135groups (n=9) received daily SC injections of the indicatedconcentrations of CG53135 on day 4 to day 9. Normal control animals(n=3) were not exposed to DSS, but did receive daily SC injections ofvehicle solution on day 4 to day 9. Animal survival was recorded on adaily basis and the experiment was concluded on day 10. Note that thedisease control and the 0.2 mg/kg CG53135 groups overlap.

DETAILED DESCRIPTION OF THE INVENTION

This invention is related in part to the discovery of novel FGF-CXnucleic acid sequences that encode polypeptides that are members of thefibroblast growth factor (“FGF”) family. As used herein the designation“FGF-CX” relates to nucleic acids, polynucleotides, proteins,polypeptides, and variants, derivatives and fragments of any of them, aswell as to antibodies that bind immunospecifically to any of theseclasses of compounds. In the present disclosure, FGF-CX polypeptides arealternatively identified by the internal accession numbers AB020858,CG53135-01 and CG53135-02.

The invention further is based on the discovery of nucleic acids thatencode polypeptides related to bone-morphogen protein-1 (“BMP-1”), tovascular endothelial growth factor (“VEGF-E”), and to platelet derivedgrowth factors (“PDGF”). These sequences are collectively referred to as“FCTRX nucleic acids” or FCTRX polynucleotides” and the correspondingencoded polypeptide is referred to as a “FCTRX polypeptide” or “FCTRXprotein.” Unless indicated otherwise, “FCTRX” is meant to refer to anyof the novel sequences disclosed herein. In addition, the polypeptidesand nucleic acids of the invention are alternately referred to hereincollectively as “PDGF D”, since they are considered to represent aheretofore unknown PDGF, i.e., one that differs from PDGF A, PDGF B andPDGF C. Furthermore, when reference is made to “PDGFXX” or “PDGF XX”wherein “X” is either the A, B, C or D, this is meant to refer tohomodimers of the particular PDGF. Alternately, when reference is madeto “PDGFXY” wherein X and Y are either the A, B, C or D, and “X” isdifferent from “Y” this is meant to refer to PDGF heterodimers.

It is shown herein that PDGF D has a high molecular weight latent form,designated p85, and a low molecular weight active form, designated p35.In the present disclosure, the FCTRX or PDGF D polypeptides arealternatively designated by the identifiers 30664188 and variationsthereof such as 30664188.0.99 or 30664188.0.331, and CG52053 andvariations thereof such as CG52053-01 and CG52053-02.

Inflammatory Bowel Disease

Inflammatory bowel disease (“IBD”) refers to a group of chronicinflammatory disorders involving the gastrointestinal tract. AlthoughIBD is diagnosed largely by exclusion, there are characteristic featuresassociated with it that allows accurate diagnosis.

Chronic IBD is sub-divided into two major groups, namely, ulcerativecolitis (“UC”) and Crohn's disease (“CD”). Clinically IBD ischaracterized by recurrent inflammatory involvement of intestinalsegments with diverse clinical manifestations. Typically UC affects therectum and extends proximally to involve part or all of the colon.Lesions are restricted to the mucosal or submucosal layers of the colonwith deeper layers unaffected except in fulminant disease. Symptomsinclude rectal bleeding, mucus containing diarrhea, abdominal pain andweight loss. CD affects the full thickness of the gut wall in both thesmall and large intestines in contrast to UC. The clinical symptoms ofUC vary according to the region affected. In general, fever, malaise,weight loss, abdominal pain and cramps are the common symptoms of CD.Full thickness bowel lesions can progress to bowel perforations andlocal abscesses, fistulas in the adjoining abdominal and pelvic organs,and fibrosis of the bowel wall with obstruction.

The etiology of UC or CD remains unknown. However, a combination offactors including abnormalities in the immune system, geneticpredisposition, environmental and psychological factors, may be ofimportance in determining the outcome of the disease.

In Europe and the United States, incidence and prevalence of CD isapproximately 1-6 and 10-100 cases per 100,000 population respectively.For UC the incidence and prevalence rates are respectively 2-10 and35-100 per 100,000. There is a slight preponderence in females overmales for contracting the disease. UC and CD affect primarilyindividuals between the ages of 15 and 35 years.

Therapeutic Options for Inflammatory Bowel Disease

Choice of therapy for IBD is dependent on pharmacodynamic considerationsthat govern drug and patient characteristics. Clinical remission (reliefof inflammatory symptoms) and mucosal healing are two vital aspects thatneed to be treated. Many of the current drugs of choice have a poorcorrelation between symptomatic relief and mucosal healing. Thus agentsthat can maintain remission as well as accomplish healing will be ofparticular interest in the management of IBD. In the past decade severaldrugs have been used in the treatment of IBD. These include conventionalsalicylates, antibiotics and corticosteroids as well as immunomodulatorsand biological response modifiers.

5-Aminosalicylates (5-ASAs)

The 5-ASAs (sulfasalazine and the sulfa-free agents) are known to alterthe immune response by down-regulating antibody secretion and lymphocytefunction, inhibit neutrophil and macrophage chemotaxis and protectintestinal epithelium by enhancing expression of heat shock proteins. Inaddition, they also inhibit the cyclooxygenase and 5-lipoxygenasepathways of arachidonic acid metabolism that may inhibit the release ofchemotactic substances (Grisham, M. B. Lancet, 1994, 344:859-861).5-ASAs are effective therapeutic agents for mild to moderate conditionsof UC. However, 5-ASAs are not the drugs of choice for IBD due to theirside effects that may include nausea, allergic reactions and reversibleoligospermia.

Antibiotics

Historically, antibiotics like metronidazole and quinolones have beenused to treat CD, although their effectiveness in ameliorating thecondition has not been well documented. The presumed effect of theseagents may be in the alteration of the bacterial flora associated withIBD. Antibiotics are not only less effective for IBD but also haveassociated side effects (anorexia, nausea, rash) and thus may not be thetreatment of choice for IBD.

Corticosteroids

Corticosteroids have been the oldest of the nonspecific but effectivetherapeutic regimen used for IBD. Corticosteroids modulate bothimmunologic and inflammatory responses and inhibit an array of leukocytefunctions such as adherence, chemotaxis, phagocytosis arachidocic acidmetabolism and eicosanoids production. Although their use in short-termtreatment of CD and UC have been shown, their efficacy in maintenancetherapy is far from satisfactory (Munkholm et al., Gut, 1994,35:360-362). The failure of corticosteroids in maintenance therapycoupled with the known detrimental side effects of this agent limittheir use in the treatment for IBD.

Immunomodulators

The thiopurine agents 6-mercaptoputine (“6-MP”) and azathioprine (“AZA”)have been used in the treatment of CD and UC as steroid sparing agents(Pearson et al.. Annals of Internal Medicine, 1995, 123:1320142). Sideeffects such as leukopenia, thrombocytopenia associated with these drugsare further complicated by the genetic predisposition of the patient.(Yates et al Ann. Intern. Med. 1997, 126:608-614). Additional sideeffects such as pancreatitis, hepatitis, nausea and rash are alsoreported.

Methotrexate has been shown to be effective in steroid-dependent CD butnot in UC The side effects of methotrexate include bone marrowsuppression, interstitial pneumonitis and neuropathy.

Cyclosporine has been effective in the treatment of both CD and UC.Cyclosporine has been particularly shown to be effective in patientswith active CD or UC that are resistant or intolerant to corticosteriods(Lichtiger et al., New England Journal of Medicine, 1994,330:1841-1845). The side effects of cyclosporin include reversible orirreversible decrease in renal function, hypertension, tremor, andseizure.

Biological Response Modifiers

The agent Infliximab, a chimeric monoclonal IgG1 antibody directedagainst TNF-α, has been effectively used in the treatment of CD.Although it is effective in maintenance therapy and healing fistulas(Present et al., New England Journal of Medicine, 1999, 340:1398-1405),side effects include delayed hypersensitivity reactions andlymphoproliferative disorders.

Fibroblast Growth Factors

The fibroblast growth factor (FGF) group of cytokines includes at least21 members that regulate diverse cellular functions such as growth,survival, apoptosis, motility and differentiation. These moleculestransduce signals via high affinity interactions with cell surfacetyrosine kinase FGF receptors (FGFRs). FGF receptors are expressed onmost types of cells in tissue culture. Dimerization of FGF receptormonomers upon ligand binding has been reported to be a requisite foractivation of the kinase domains, leading to receptor transphosphorylation. FGF receptor-1 (FGFR-1), which shows the broadestexpression pattern of the four FGF receptors, contains at least seventyrosine phosphorylation sites. A number of signal transductionmolecules are affected by binding with different affinities to thesephosphorylation sites.

Previously described members of the FGF family regulate diverse cellularfunctions such as growth, survival, apoptosis, motility anddifferentiation (Szebenyi & Fallon (1999) Int. Rev. Cytol. 185, 45-106).These molecules transduce signals intracellularly via high affinityinteractions with cell surface tyrosine kinase FGF receptors (FGFRs),four of which have been identified to date (Xu et al. (1999) Cell TissueRes. 296, 33-43; Klint & Claesson-Welsh (1999) Front. Biosci. 4,165-177). These FGF receptors are expressed on most types of cells intissue culture. Dimerization of FGF receptor monomers upon ligandbinding has been reported to be a requisite for activation of the kinasedomains, leading to receptor trans phosphorylation. FGF receptor-1(FGFR-1), which shows the broadest expression pattern of the four FGFreceptors, contains at least seven tyrosine phosphorylation sites. Anumber of signal transduction molecules are affected by binding withdifferent affinities to these phosphorylation sites.

FGFs also bind, albeit with low affinity, to heparin sulfateproteoglycans (HSPGs) present on most cell surfaces and extracellularmatrices (ECM). Interactions between FGFs and HSPGs serve to stabilizeFGF/FGFR interactions, and to sequester FGFs and protect them fromdegradation (Szebenyi. & Fallon (1999)). Due to its growth-promotingcapabilities, one member of the FGF family, FGF-7, is currently inclinical trials for the treatment of chemotherapy-induced mucositis(Danilenko (1999) Toxicol. Pathol. 27, 64-71).

In addition to participating in normal growth and development, knownFGFs have also been implicated in the generation of pathological states,including cancer (Basilico & Moscatelli (1992) Adv. Cancer Res. 59,115-165). FGFs may contribute to malignancy by directly enhancing thegrowth of tumor cells. For example, autocrine growth stimulation throughthe co-expression of FGF and FGFR in the same cell leads to cellulartransformation (Matsumoto-Yoshitomi, et al., (1997) Int. J. Cancer 71,442-450). Likewise, the constitutive activation of FGFR via mutation orrearrangement leads to uncontrolled proliferation (Lorenzi, et al.,(1996) Proc. Natl. Acad. Sci. USA. 93, 8956-8961; Li, et al., (1997)Oncogene 14, 1397-1406). Furthermore, some FGFs are angiogenic (Gerwins,et al., (2000) Crit. Rev. Oncol. Hematol. 34, 185-194). Such FGFs maycontribute to the tumorigenic process by facilitating the development ofthe blood supply needed to sustain tumor growth. Not surprisingly, atleast one FGF is currently under investigation as a potential target forcancer therapy (Gasparini (1999) Drugs 58, 17-38).

Expression of FGFs and their receptors in the brains of perinatal andadult mice has been examined. Messenger RNA all FGF genes, with theexception of FGF-4, is detected in these tissues. FGF-3, FGF-6, FGF-7and FGF-8 genes demonstrate higher expression in the late embryonicstages than in postnatal stages, suggesting that these members areinvolved in the late stages of brain development. In contrast,expression of FGF-1 and FGF-5 increased after birth. In particular,FGF-6 expression in perinatal mice has been reported to be restricted tothe central nervous system and skeletal muscles, with intense signals inthe developing cerebrum in embryos but in cerebellum in 5-day-oldneonates. FGF-receptor (FGFR)-4, a cognate receptor for FGF-6,demonstrate similar spatiotemporal expression, suggesting that FGF-6 andFGFR-4 plays significant roles in the maturation of nervous system as aligand-receptor system. According to Ozawa et al., these resultsstrongly suggest that the various FGFs and their receptors are involvedin the regulation of a variety of developmental processes of brain, suchas proliferation and migration of neuronal progenitor cells, neuronaland glial differentiation, neurite extensions, and synapse formation.

Glia-activating factor (“GAF”), another FGF family member, is aheparin-binding growth factor that was purified from the culturesupernatant of a human glioma cell line. See, Miyamoto et al., 1993, MolCell Biol 13(7): 4251-4259. GAF shows a spectrum of activity slightlydifferent from those of other known growth factors, and is designated asFGF-9. The human FGF-9 cDNA encodes a polypeptide of 208 amino acids.Sequence similarity to other members of the FGF family was estimated tobe around 30%. Two cysteine residues and other consensus sequences foundin other family members were also well conserved in the FGF-9 sequence.FGF-9 was found to have no typical signal sequence in its N terminuslike those in acidic FGF and basic FGF.

Acidic FGF and basic FGF are known not to be secreted from cells in aconventional manner. However, FGF-9 was found to be secreted efficientlyfrom cDNA-transfected COS cells despite its lack of a typical signalsequence. It could be detected exclusively in the culture medium ofcells. The secreted protein lacked no amino acid residues at the Nterminus with respect to those predicted by the cDNA sequence, exceptthe initiation methionine. The rat FGF-9 cDNA was also cloned, and thestructural analysis indicated that the FGF-9 gene is highly conserved.

Platelet Derived Growth Factors

Polypeptide growth factors exerting effects in a variety of tissues havebeen described. Among these growth factors are bone morphogeneticprotein-1 (“BMP-1”), vascular endothelial growth factor (VEGF), andplatelet-derived growth factor (“PDGF”).

Multiple effects have been attributed to BMP-1. For example, BMP-1 iscapable of inducing formation of cartilage in vivo. BMP1 is alsoidentical to purified procollagen C proteinase (“PCP”), a secretedcalcium-dependent metalloprotease that has been reported to be requiredfor cartilage and bone formation. BMP-1 cleaves the C-terminalpropeptides of procollagen I, II, and III and its activity is increasedby the procollagen C-endopeptidase enhancer protein.

Vascular endothelial growth factor (“VEGF”) polypeptides have beenreported to act as mitogens primarily for vascular endothelial cells.The specificity for vascular endothelial cells contrasts VEGFpolypeptides from other polypeptide mitogens, such as basic fibroblastgrowth factor and platelet-derived growth factors, which are active on awider range of cell types.

VEGF has also been reported to affect tumor angiogenesis. For example,VEGF has been shown to stimulate the elongation, network formation, andbranching of nonproliferating endothelial cells in culture that aredeprived of oxygen and nutrients.

The platelet derived growth factor (“PDGF”) family currently consists ofat least 3 distinct genes, PDGF A, PDGF B, and PDGF C whose geneproducts selectively signal through two PDGFRs to regulate diversecellular functions. PDGF A, PDGF B, and PDGF C dimerize in solution toform homodimers, as well as the heterodimer.

Expression of RNA encoding the PDGF A and PDGF B subunits of has beenreported in vascular tissues involved in atherosclerosis. PDGF A andPDGF B mRNA have been reported to be present in mesenchymal-appearingintimal cells and endothelial cells, respectively, of atheroscleroticplaques. In addition, PDGF receptor mRNA has also been localizedpredominantly in plaque intimal cells.

The PDGF B is related to the transforming gene (v-sis) of simian sarcomavirus. The PDGF B has also been reported to be mitogen for cells ofmesenchymal origin. The PDGF B has in addition been implicated inautocrine growth stimulation in the pathologic proliferation ofendothelial cells characteristically found in glioblastomas. PDGF hasalso been reported to promote cellular proliferation and inhibitsapoptosis.

FGF-CX

The present invention is related to a novel human FGF as well as itscorresponding cDNA. The protein product of this gene has been shown toexhibit growth stimulatory and growth promoting properties.

The nucleotide sequence and translated polypeptide sequence ofFibroblast Growth Factor-CX (“FGF-CX,” also referred to as AB020858) ispresented in Table 1 (see Example 1; see also disclosure in U.S. Ser.No. 60/145,899, filed Jul. 27, 1999, U.S. Ser. No. 09/494585, filed Jan.31, 2000 and U.S. Ser. No. 09/609,543, filed Jul. 3, 2000, all of whichare incorporated herein by reference in their entireties). The start andstop codons are shown in bold.

TABLE 1 Nucleotide (SEQ ID NO:1) and Protein (SEQ ID NO:2) Sequence ofFibroblast Growth Factor-CX (FGF-CX)   1ATGGCTCCCTTAGCCGAAGTCGGGGGCTTTCTGGGCGGCCTGGAGMetAlaProLeuAlaGluValGlyGlyPheLeuGlyGlyLeuGlu  46GGCTTGGGCCAGCAGGTGGGTTCGCATTTCCTGTTGCCTCCTGCCGlyLeuGlyGlnGlnValGlySerHisPheLeuLeuProProAla  91GGGGAGCGGCCGCCGCTGCTGGGCGAGCGCAGGAGCGCGGCGGAGGlyGluArgProProLeuLeuGlyGluArgArgSerAlaAlaGlu 136CGGAGCGCGCGCGGCGGGCCGGGGGCTGCGCAGCTGGCGCACCTGArgSerAlaArgGlyGlyProGlyAlaAlaGlnLeuAlaHisLeu 181CACGGCATCCTGCGCCGCCGGCAGCTCTATTGCCGCACCGGCTTCHisGlyIleLeuArgArgArgGlnLeuTyrCysArgThrGlyPhe 226CACCTGCAGATCCTGCCCGACGGCAGCGTGCAGGGCACCCGGCAGHisLeuGlnIleLeuProAspGlySerValGlnGlyThrArgGln 271GACCACAGCCTCTTCGGTATCTTGGAATTCATCAGTGTGGCAGTGAspHisSerLeuPheGlyIleLeuGluPheIleSerValAlaVal 316GGACTGGTCAGTATTAGAGGTGTGGACAGTGGTCTCTATCTTGGAGlyLeuValSerIleArgGlyValAspSerGlyLeuTyrLeuGly 361ATGAATGACAAAGGAGAACTCTATGGATCAGAGAAACTTACTTCCMetAsnAspLysGlyGluLeuTyrGlySerGluLysLeuThrSer 406GAATGCATCTTTAGGGAGCAGTTTGAAGAGAACTGGTATAACACCGluCysIlePheArgGluGlnPheGluGluAsnTrpTyrAsnThr 451TATTCATCTAACATATATAAACATGGAGACACTGGCCGCAGGTATTyrSerSerAsnIleTyrLysHisGlyAspThrGlyArgArgTyr 496TTTGTGGCACTTAACAAAGACGGAACTCCAAGAGATGGCGCCAGGPheValAlaLeuAsnLysAspGlyThrProArgAspGlyAlaArg 541TCCAAGAGGCATCAGAAATTTACACATTTCTTACCTAGACCAGTGSerLysArgHisGlnLysPheThrHisPheLeuProArgProVal 586GATCCAGAAAGAGTTCCAGAATTGTACAAGGACCTACTGATGTACAspProGluArgValProGluLeuTyrLysAspLeuLeuMetTyr 631 ACT* (SEQ ID NO:1) Thr(SEQ ID NO:2)

Included in the invention is a nucleotide sequence (SEQ ID NO:1)encoding a novel fibroblast growth factor designated fibroblast growthfactor-20X (FGF-CX) (see Table 1; SEQ ID NO:1). This coding sequence wasidentified in human genomic DNA sequences. The disclosed DNA sequencehas 633 bases that encode a polypeptide predicted to have 211 amino acidresidues (Table 1; SEQ ID NO:2). The predicted molecular weight ofFGF-CX, based on the sequence shown in Table 1 and SEQ ID NO:2, is23498.4 Da.

The FGF-CX nucleic acid sequence was used as a query nucleotide sequencein a BLASTN search to identify related nucleic acid sequences. TheFGF-CX nucleotide sequence has a high similarity to murine fibroblastgrowth factor 9 (“FGF-9”) (392 of 543 bases identical, or 72%; GenBankAccession Number S82023) and to human DNA encoding glia activatingfactor (GAP) (385 of 554 bases identical, or 69%; GenBank AccessionNumber E05822, also termed FGF-9). In addition, FGF-CX was found to havea comparable degree of identity (311 of 424 bases identical, or 73%) toa GAF sequence (SEQ ID NO:5) disclosed by Naruo et al. in JapanesePatent: JP 1993301893 entitled “Glia-Activating Factor And ItsProduction”.

To verify that the open reading frame (ORF) identified by genomic miningwas correct, PCR amplification was used to obtain a cDNA correspondingto the predicted genomic clone. The nucleotide sequence of the obtainedproduct precisely matches that of the predicted gene (see Example 2).

The protein encoded by the cDNA is most closely related to XenopusFGF-20X (designated XFGF-CX or XFGF-20X herein), as well as to humanFGF-9 and human FGF-16 (80%, 70% and 64% amino acid identity,respectively). Based on the strong homology with XFGF-CX, the geneidentified in the present disclosure is believed to represent its humanortholog, and is named FGF-CX herein.

A BLASTP analyses of the polypeptide of SEQ ID NO:2 shows that the first208 amino acids of the FGF-CX polypeptide sequence (SEQ ID NO:2) alignswith a human FGF-9. See, e.g., SWISSPROT Accession Number P31371 forGlia-Activating Factor Precursor (GAF) (Fibroblast Growth Factor-9);Miyamoto et al. 1993 Mol. Cell. Biol. 13:4251-4259; and Naruo et al.1993 J. Biol. Chem. 268:2857-2864. BLASTX analysis shows that the first208 amino acids of the FGF-CX polypeptide (SEQ ID NO:2 aligns with themouse FGF-9 and rat FGF-9 sequences. See, e.g., SWISSPROT AccessionNumber P54130 for Glia-Activating Factor Precursor (GAF) (FibroblastGrowth Factor-9), Santos-Ocampo et al., 1996 J. Biol. Chem.271:1726-1731, for mouse FGF-9; and SWISSPROT Accession Number P36364Glia-Activating Factor Precursor (GAF) (Fibroblast Growth Factor-9)(FGF-9), Miyamoto, 1993 Mol. Cell. Biol. 13:4251-4259, for rat FGF-9.

The full length FGF-CX polypeptide (SEQ ID NO:2) was also aligned byBLASTX with Xenopus XFGF-CX (See, Koga et al., 1999 Biochem Biophys ResCommun 261(3):756-765). It was found that FGF-CX has 170 of 211 (80%)identical residues, and 189 of 211 (89%) positive residues compared withXenopus XFGF-CX. The deduced 208 amino acid sequence of the XFGF-CX openreading frame contains a motif characteristic of the FGF family. XFGF-CXhas a 73.1% overall similarity to XFGF-9 but differs from XFGF-9 in itsamino-terminal region (33.3% similarity). This resembles the similarityseen for the presently disclosed SEQ ID NO:2 with respect to variousmammalian FGF-9 and FGF-16 sequences, including human (see above).

FGF-CX lacks a classical amino-terminal signal sequence as predicted byPSORT (Nakai & Kanehisa (1992) Genomics 14, 897-911) and SIGNALP(Nielsen et al. (1997) Protein Eng. 10, 1-6) computer algorithms, justas found for some of its closest human family members (e.g. FGF-9 andFGF-16). Nonetheless, both FGF-9 and FGF-16 are secreted(Matsumoto-Yoshitomi et al. (1997) Int. J. Cancer 71, 442-450; Miyake etal. (1998) Biochem. Biophys. Res. Comm. 243, 148-152; Miyakawa et al.(1999) J. Biol. Chem. 274, 29352-29357; Revest et al. (2000) J Biol.Chem. 275, 8083-8090). To determine whether FGF-CX is also secreted, thecDNA encoding the full length FGF-CX protein was subcloned into amammalian expression vector designated pFGF-CX. The protein expressedwhen human embryonic kidney 293 cells are transfected with this vectoris found in the conditioned medium, and exhibits a band detected by anantibody to a C-terminal V5 epitope, with an apparent molecular weightin a Western blot of ˜27 kDa (FIG. 4). An additional portion of theexpressed protein is released from sequestration on the 293 cells bytreatment with a substance that inhibits interaction with heparinsulfate proteoglycan (HSPG). The protein released in this way alsoexhibits a similar Western blot pattern (FIG. 4). Similarly when theprotein is expressed in HEK293 cells from a recombinant plasmidincorporating an Ig Kappa signal sequence, a band is detected by Westernblot with an apparent molecular weight of approximately 34 kDa (FIG. 1,Example 4).

FCTR1

A polynucleotide of the invention includes the nucleic acid of FCTR1(also referred to as clone 30664188.0.99). FCTR1 is 1828 nucleotides inlength. The nucleotide sequence of FCTR1 (also referred to as30664188.0.99 or PDGFD) is reported in Table 2 (SEQ ID NO:3). The clonewas orginally obtained from RNA from pituitary gland tissues is alsopresent in RNA from human uterine microvascular endothelial cells(Clonetics, San Diego, Calif.), human erythroleukemia cells (ATCC,Manassas, Va.), thyroid, small intestine, lymphocytes, adrenal gland andsalivary gland. The untranslated regions upstream of the start site anddownstream of the stop codon are underlined, and the start and stopcodons are shown in bold.

TABLE 2 Nucleotide (SEQ ID NO:3) and Protein (SEQ ID NO:4) Sequence ofFCTR1 Translated Protein—Frame: 2—Nucleotide 182 to 1291    1CTAAAAAATATGTTCTCTACAACACCAAGGCTCATTAAAATATTT   46TAAATATTAATATACATTTCTTCTGTCAGAAATACATAAAACTTT   91ATTATATCAGCGCAGGGCGGCGCGGCGTCGGTCCCGGGAGCAGAA  136CCCGGCTTTTTCTTGGAGCGACGCTGTCTCTAGTCGCTGATCCCA  181 AATGCACCGGCTCATCTTTGTCTACACTCTAATCTGCGCAAACTT MetHisArgLeuIlePheValTyrThrLeuIleCysAlaAsnPh  226TTGCAGCTGTCGGGACACTTCTGCAACCCCGCAGAGCGCATCCATeCysSerCysArgAspThrSerAlaThrProGlnSerAlaSerIl  271CAAAGCTTTGCGCAACGCCAACCTCAGGCGAGATGAGAGCAATCAeLysAlaLeuArgAsnAlaAsnLeuArgArgAspGluSerAsnHi  316CCTCACAGACTTGTACCGAAGAGATGAGACCATCCAGGTGAAAGGsLeuThrAspLeuTyrArgArgAspGluThrIleGlnValLysGl  361AAACGGCTACGTGCAGAGTCCTAGATTCCCGAACAGCTACCCCAGyAsnGlyTyrValGlnSerProArgPheProAsnSerTyrProAr  406GAACCTGCTCCTGACATGGCGGCTTCACTCTCAGGAGAATACACGgAsnLeuLeuLeuThrTrpArgLeuHisSerGlnGluAsnThrAr  451GATACAGCTAGTGTTTGACAATCAGTTTGGATTAGAGGAAGCAGAgIleGlnLeuValPheAspAsnGlnPheGlyLeuGluGluAlaGl  496AAATGATATCTGTAGGTATGATTTTGTGGAAGTTGAAGATATATCuAsnAspIleCysArgTyrAspPheValGluValGluAspIleSe  541CGAAACCAGTACCATTATTAGAGGACGATGGTGTGGACACAAGGArGluThrSerThrIleIleArgGlyArgTrpCysGlyHisLysGl  586AGTTCCTCCAAGGATAAAATCAAGAACGAACCAAATTAAAATCACuValProProArgIleLysSerArgThrAsnGlnIleLysIleTh  631ATTCAAGTCCGATGACTACTTTGTGGCTAAACCTGGATTCAAGATrPheLysSerAspAspTyrPheValAlaLysProGlyPheLysIl  676TTATTATTCTTTGCTGGAAGATTTCCAACCCGCAGCAGCTTCAGAeTyrTyrSerLeuLeuGluAspPheGlnProAlaAlaAlaSerGl  721GACCAACTGGGAATCTGTCACAAGCTCTATTTCAGGGGTATCCTAuThrAsnTrpGluSerValThrSerSerIleSerGlyValSerTy  766TAACTCTCCATCAGTAACGGATCCCACTCTGATTGCGGATGCTCTrAsnSerProSerValThrAspProThrLeuIleAlaAspAlaLe  811GGACAAAAAAATTGCAGAATTTGATACAGTGGAAGATCTGCTCAAuAspLysLysIleAlaGluPheAspThrValGluAspLeuLeuLy  856GTACTTCAATCCAGAGTCATGGCAAGAAGATCTTGAGAATATGTAsTyrPheAsnProGluSerTrpGlnGluAspLeuGluAsnMetTy  901TCTGGACACCCCTCGGTATCGAGGCAGGTCATACCATGACCGGAArLeuAspThrProArgTyrArgGlyArgSerTyrHisAspArgLy  946GTCAAAAGTTGACCTGGATAGGCTCAATGATGATGCCAAGCGTTAsSerLysValAspLeuAspArgLeuAsnAspAspAlaLysArgTy  991CAGTTGCACTCCCAGGAATTACTCGGTCAATATAAGAGAAGAGCTrSerCysThrProArgAsnTyrSerValAsnIleArgGluGluLe 1036GAAGTTGGCCAATGTGGTCTTCTTTCCACGTTGCCTCCTCGTGCAuLysLeuAlaAsnValValPhePheProArgCysLeuLeuValGl 1081GCGCTGTGGAGGAAATTGTGGCTGTGGAACTGTCAACTGGAGGTCnArgCysGlyGlyAsnCysGlyCysGlyThrValAsnTrpArgSe 1126CTGCACATGCAATTCAGGGAAAACCGTGAAAAAGTATCATGAGGTrCysThrCysAsnSerGlyLysThrValLysLysTyrHisGluVa 1171ATTACAGTTTGAGCCTGGCCACATCAAGAGGAGGGGTAGAGCTAAlLeuGlnPheGluProGlyHisIleLysArgArgGlyArgAlaLy 1216GACCATGGCTCTAGTTGACATCCAGTTGGATCACCATGAACGATGsThrMetAlaLeuValAspIleGlnLeuAspHisHisGluArgCy 1261TGATTGTATCTGCAGCTCAAGACCACCTCGATAA GAGAATGTGCASAspCysIleCysSerSerArgProProArg 1306CATCCTTACATTAAGCCTGAAAGAACCTTTAGTTTAAGGAGGGTG 1352AGATAAGAGACCCTTTTCCTACCAGCAACCAAACTTACTACTAGC 1396CTGCAATGCAATGAACACAAGTGGTTGCTGAGTCTCAGCCTTGCT 1441TTGTTAATGCCATGGCAAGTAGAAAGGTATATCATCAACTTCTAT 1486ACCTAAGAATATAGGATTGCATTTAATAATAGTGTTTGAGGTTAT 1531ATATGCACAAACACACACAGAAATATATTCATGTCTATGTGTATA 1576TAGATCAAATGTTTTTTTTGGTATATATAACCAGGTACACCAGAG 1621CTTACATATGTTTGAGTTAGACTCTTAAAATCCTTTGCCAAAATA 1666AGGGATGGTCAAATATATGAAACATGTCTTTAGAAAATTTAGGAG 1711ATAAATTTATTTTTAAATTTTGAAACACAAAACAATTTTGAATCT 1756TGCTCTCTTAAAGAAAGCATCTTGTATATTAAAAATCAAAAGATG 1801AGGCTTTCTTACATATACATCTTAGTTG

Nucleotides 182 to 1292 of SEQ ID NO:3 encode a 370 amino acid protein(SEQ ID NO:4) that includes sequences characteristic of secretedproteins. The sequence of the encoded protein, which is also referred toherein as “FCTR1 protein,” “30664188.0.99 protein,” “30664188.0.99,”“PDGFD,” or “human PDGFD” is presented in Table 2. The predictedmolecular weight of the 30664188.0.99 protein is 42847.8 daltons with apI of 7.88.

BLASTN and BLASTP analyses indicate the 30664188.0.99 polypeptide has asimilarity to human vascular endothelial growth factor E (VEGF-E), aswell as to VEGF-E from other vertebrate species. For example, there is a44% identity to human secretory growth factor-like protein (VEGF-E, orfallotein; Acc. No.: AAF00049 which references GenBank-ID: AF091434 forthe nucleotide sequence). An alignment of the amino acid sequence of the30664188.0.99 polypeptide with that of VEGF-E is shown in FIG. 1. BLASTPanalyses also indicate that FCTR1 is related to human PDGF C, PDGF B,and PDGF A (42%, 27%, and 25% overall amino acid identity, respectively)

PFAM and PROSITE analyses indicte that 30664188.0.99 polypeptide aminoacid sequence conatains a PDGF domain (aa 272-362) and a N-linkedglycosylation site (residue 276).

The 30664188.0.99 polypeptide amino acid sequence shows similarity tothe sequence of human procollagen C-endopeptidase (bone morphogeneticprotein-1; BMP-1; PIR-ID:A58788), which is a polypeptide of 823residues. Residues 54 to 169 of the 30664188.0.99 polypeptide show30-41% identity over three segments of the BMP-1 polypeptide. The30664188.0.99 polypeptide also shows a similar degree of identity is toBMP-1 from Xenopus laevis (ACC NO:P98070), which is a 707 residueprotein. The latter protein may act as a zinc protease in promotingcartilage and bone formation (Wozney et al., Science 242: 1528-34,1988).

The 30664188.0.99 polypeptide is also related to other growth factors.For example, it shows 42% identity and 59% similarity to chicken spinalcord-derived growth factor (TREMBLNEW-ACC:BAB03265), 42% identity and59% identity to human secretory growth factor-like protein fallotein(SPTREMBL-ACC:Q9UL22), 42% identity and 39% similarity to humanplatelet-derived growth factor C (TREMBLNEW-ACC:AAF80597), and 39%identity and 59% similarity to mouse fallotein (SPTREMBL-ACC:Q9QY71).

The homologies discussed above identify the 30664188.0.99 polypeptide asa member of the BMP-1/VEGF-E/PDGF protein family. BMP-1 proteins includean EGF-like domain, three CUB domains, and PDGF/VEGF domains. BMP-1proteins are also members of the astacin subfamily.

SignalP and PSORT analyses predict that the amino acid sequence for30664188.0.99 includes a cleavable amino terminal signal peptide with acleavage site between positions 23 and 24 (TSA-TP). The protein is mostlikely secreted and localized outside of the cell. The InterPro softwareprogram predicts the presence of a CUB domain in 30664188.0.99 fromresidue 53 to residue 167, a PDGF domain spanning residues 272-306 and350-362, and a metallothionein domain from residue 302 to residue 365. AFCTR1 polypeptide of the invention includes a polypeptide having one,two, three, or four of these domains, or a combination thereof.

A FCTR1polypeptide of the invention includes a mature form of a FCTR1polypeptide that includes amino acids 24-370 of SEQ ID NO:4. Thesesequences are also encoded in a construct encoded by clone30664188.0.m99, which is described in more detail below. Also within theinvention are nucleic acids encoding FCTRX polypeptide fragments thatinclude amino acid sequences 247-370, 247-338, or 339-370, or theirvariant forms. In some embodiments, the fragments stimulateproliferation of cells. Also within the invention are the FCTRXpolypeptide fragments, or their variants, homologs or analogs encoded bythese nucleic acids.

FCTR2 Nucleic Acids and Polypeptides

A polynucleotide of the invention includes the nucleic acid sequence ofFCTR2 (also referred to as clone 30664188.0.331). FCTR2 is 1587nucleotides in length and was originally isolated from RNA frompituitary gland tissues. The nucleotide sequence of FCTR2 is shown inTable 3 (SEQ ID NO:5). The untranslated regions upstream of the startsite and downstream of the stop codon are underlined, and the start andstop codons are shown in bold.

TABLE 3 Nucleotide (SEQ ID NO:5) and Protein (SEQ ID NO:6) Sequence ofFCTR2 Translated Protein—Frame: 3—Nucleotide 540 to 935    1AGAGGCTCTCAAATTAGATCAAGAAATGCCTTTAACAGAAGTGAA   46GAGTGAACCTGCTCCTGACATGGCGGCTTCACTCTCAGGAGAATA   91CACGGATACAGCTAGTGTTTGACAATCAGTTTGGATTAGAGGAAG  136CAGAAAATGATATCTGTAGGTATGATTTTGTGGAAGTTGAAGATA  181TATCCGAAACCAGTACCATTATTAGAGGACGATGGTGTGGACACA  226AGGAAGTTCCTCCAAGGATAAAATCAAGAACGAACCAAATTAAAA  271TCACATTCAAGTCCGATGACTACTTTGTGGCTAAACCTGGATTCA  316AGATTTATTATTCTTTGCTGGAAGATTTCCAACCCGCAGCAGCTT  361CAGAGACCAACTGGGAATCTGTCACAAGCTCTATTTCAGGGGTAT  406CCTATAACTCTCCATCAGTAACGGATCCCACTCTGATTGCGGATG  451CTCTGGACAAAAAAATTGCAGAATTTGATACAGTGGAAGATCTGC  496TCAAGTACTTCAATCCAGAGTCATGGCAAGAAGATCTTGAGAATA                                            M  541TGTATCTGGACACCCCTCGGTATCGAGGCAGGTCATACCATGACCetTyrLeuAspThrProArgTyrArgGlyArgSerTyrHisAspA  586GGAAGTCAAAAGTTGACCTGGATAGGCTCAATGATGATGCCAAGCrgLysSerLysValAspLeuAspArgLeuAsNAspAspAlaLysA  631GTTACAGTTGCACTCCCAGGAATTACTCGGTCAATATAAGAGAAGrgTyrSerCysThrProArgAsnTyrSerValAsnIleArgGluG  676AGCTGAAGTTGGCCAATGTGGTCTTCTTTCCACGTTGCCTCCTCGluLeuLysLeuAlaAsnValValPhePheProArgCysLeuLeuV  721TGCAGCGCTGTGGAGGAAATTGTGGCTGTGGAACTGTCAACTGGAalGlnArgCysGlyGlyAsnCysGlyCysGlyThrValAsnTrpA  766GGTCCTGCACATGCAATTCAGGGAAAACCGTGAAAAAGTATCATGrgSerCysThrCysAsnSerGlyLysThrValLysLysTyrHisG  811AGGTATTACAGTTTGAGCCTGGCCACATCAAGAGGAGGGGTAGAGluValLeuGlnPheGluProGlyHisIleLysArgArgGlyArgA  856CTAAGACCATGGCTCTAGTTGACATCCAGTTGGATCACCATGAAClaLysThrMetAlaLeuValAspIleGlnLeuAspHisHisGluA  901GATGTGATTGTATCTGCAGCTCAAGACCACCTCGATAA GAGAATGRgCysAspCysIleCysSerSerArgProProArg  946TGCACATCCTTACATTAAGCCTGAAAGAACCTTTAGTTTAAGGAG  991GGTGAGATAAGAGACCCTTTTCCTACCAGCAACCAAACTTACTAC 1036TAGCCTGCAATGCAATGAACACAAGTGGTTGCTGAGTCTCAGCCT 1081TGCTTTGTTAATGCCATGGCAAGTAGAAAGGTATATCATCAACTT 1126CTATACCTAAGAATATAGGATTGCATTTAATAATAGTGTTTGAGG 1171TTATATATGCACAAACACACACAGAAATATATTCATGTCTATGTG 1216TATATAGATCAAATGTTTTTTTTGGTATATATAACCAGGTACACC 1261AGAGCTTACATATGTTTGAGTTAGACTCTTAAAATCCTTTGCCAA 1306AATAAGGGATGGTCAAATATATGAAACATGTCTTTAGAAAATTTA 1351GGAGATAAATTTATTTTTAAATTTTGAAACACAAAACAATTTTGA 1396ATCTTGCTCTCTTAAAGAAAGCATCTTGTATATTAAAAATCAAAA 1441GATGAGGCTTTCTTACATATACATCTTAGTTGATTATTAAAAAAG 1486GAAAAATATGGTTTCCAGAGAAAAGGCCAATACCTAAGCATTTTT 1531TCCATGAGAAGCACTGCATACTTACCTATGTGGACTATAATAACC 1576 TGTCTCCAAAAC

Clone 30664188.0.331 includes an open reading frame from nucleotides 540to 936. The open reading frame encodes a polypeptide of 132 amino acids(SEQ ID NO:6). The encoded polypeptide is referred to herein as the“30664188.0.331 protein” or the “30664188.0.331 polypeptide”. Thepredicted amino acid sequence of the 30664188.0.331 nucleic acidsequence is shown in Table 3 (SEQ ID NO:6).

Nucleotides 50 to 1472 of clone 30664188.0.331 are 100% identical tonucleotides 406-1828 of clone 30664188.0.99. The 132 amino acids of theclone 30664188.0.331 protein are 100% identical to the carboxy-terminalregion of the protein sequence of 30664188.0.99. Thus, the nucleic acidsof clones 30664188.0.99 and 30664188.0.331 are therefore related assplice variants of a common gene.

The 30664188.0.331 protein shows similarity to human growth factor FIGF(c-fos-induced growth factor; ptnr:SPTREMBL-ACC:O43915), a member of theplatelet-derived growth factor/vascular endothelial growth factor(PDGF/VEGF) family, and to rat vascular endothelial growth factor D(ptnr:SPTREMBL-ACC:O35251).

FCTR3 Acids and Polypeptides

A FCTR3 (also referred to within the specification as PDGFD or murinePDGFD or mPDGFD) nucleic acid and polypeptide according to the inventionincludes the nucleic acid and encoded polypeptide sequence shown inTable 4 (SEQ ID NO:7 and 8). The start and stop codons are shown inbold. The FCTR3 nucleic acid sequence was identified from a murine brainlibrary. The predicted open reading frame codes for a 370 amino acidlong secreted protein. The FCTR3 has a predicted molecular weight of42,808 daltons and a pI of 7.53. Protein structure analysis using PFAMand PROSITE identified the core PDGF domain within the FCTR3 polypeptidesequence.

TABLE 4 Nucleotide (SEQ ID NO:7) and Protein (SEQ ID NO:8) Sequence ofFCTR3    1ATGCAACGGCTCGTTTTAGTCTCCATTCTCCTGTGCGCGAACTTTAGCTGCTATCCGGACACTTTTGCGACTCCGCAGAGM  Q  R  L  V  L  V  S  I  L  L  C  A  N  F  S  C  Y  P  D  T  F  A  T  P  Q  R  81AGCATCCATCAAAGCTTTGCGCAATGCCAACCTCAGGAGAGATGAGAGCAATCACCTCACAGACTTGTACCAGAGAGAGG A  S  I  K  A  L  R  N  A  N  L  R  R  D  E  S  N  H  L  T  D  L  Y  Q  R  E  E 161AGAACATTCAGGTGACAAGCAATGGCCATGTGCAGAGTCCTCGCTTCCCGAACAGCTACCCAAGGAACCTGCTTCTGACA  N  I  Q  V  T  S  N  G  H  V  Q  S  P  R  F  P  N  S  Y  P  R  N  L  L  L  T 241TGGTGGCTCCGTTCCCAGGAGAAAACACGGATACAACTGTCCTTTGACCATCAATTCGGACTAGAGGAAGCAGAAAATGAW  W  L  R  S  Q  E  K  T  R  I  Q  L  S  F  D  H  Q  F  G  L  E  E  A  E  N  D 321CATTTGTAGGTATGACTTTGTGGAAGTTGAAGAAGTCTCAGAGAGCAGCACTGTTGTCAGAGGAAGATGGTGTGGCCACA I  C  R  Y  D  F  V  E  V  E  E  V  S  E  S  S  T  V  V  R  G  R  W  C  G  H  K 401AGGAGATCCCTCCAAGGATAACGTCAAGAACAAACCAGATTAAAATCACATTTAAGTCTGATGACTACTTTGTGGCAAAA  E  I  P  P  R  I  T  S  R  T  N  Q  I  K  I  T  F  K  S  D  D  Y  F  V  A  K 481CCTGGATTCAAGATTTATTATTCATTTGTGGAAGATTTCCAACCGGAAGCAGCCTCAGAGACCAACTGGGAATCAGTCACP  G  F  K  I  Y  Y  S  F  V  E  D  F  Q  P  E  A  A  S  E  T  N  W  E  S  V  T 561AAGCTCTTTCTCTGGGGTGTCCTATCACTCTCCATCAATAACGGACCCCACTCTCACTGCTGATGCCCTGGACAAAACTG S  S  F  S  G  V  S  Y  H  S  P  S  I  T  D  P  T  L  T  A  D  A  L  D  K  T  V 641TCGCAGAATTCGATACCGTGGAAGATCTACTTAAGCACTTCAATCCAGTGTCTTGGCAAGATGATCTGGAGAATTTGTAT  A  E  F  D  T  V  E  D  L  L  K  H  F  N  P  V  S  W  Q  D  D  L  E  N  L  Y 721CTGGACACCCCTCATTATAGAGGCAGGTCATACCATGATCGGAAGTCCAAAGTGGACCTGGACAGGCTCAATGATGATGTL  D  T  P  H  Y  R  G  R  S  Y  H  D  R  K  S  K  V  D  L  D  R  L  N  D  D  V 801CAAGCGTTACAGTTGCACTCCCAGGAATCACTCTGTGAACCTCAGGGAGGAGCTGAAGCTGACCAATGCAGTCTTCTTCC K  R  Y  S  C  T  P  R  N  H  S  V  N  L  R  E  E  L  K  L  T  N  A  V  F  F  P 881CACGATGCCTCCTCGTGCAGCGCTGTGGTGGCAACTGTGGTTGCGGAACTGTCAACTGGAAGTCCTGCACATGCAGCTCA  R  C  L  L  V  Q  R  C  G  G  N  C  G  C  G  T  V  N  W  K  S  C  T  C  S  S 961GGGAAGACAGTGAAGAAGTATCATGAGGTATTGAAGTTTGAGCCTGGACATTTCAAGAGAAGGGGCAAAGCTAAGAATATG  K  T  V  K  K  Y  H  E  V  L  K  F  E  P  G  H  F  K  R  R  G  K  A  K  N  M1041GGCTCTTGTTGATATCCAGCTGGATCATCATGAGCGATGTGACTGTATCTGCAGCTCAAGACCACCTCGATAA A  L  V  D  I  Q  L  D  H  H  E  R  C  D  C  I  C  S  S  R  P  P  RFCTR4 Nucleic Acids and Polypeptides

A FCTR4 (also referred to within the specification as PDGFD or murinePDGFD or mPDGFD) nucleic acid and polypeptide according to the inventionincludes the nucleic acid and encoded polypeptide sequence shown inTable 5 (SEQ ID NO:9 and 10). The start and stop codons are shown inbold. The FCTR4 nucleic acid sequence was identified from a murine brainlibrary and is a splice variant of FCTR3. FCTR4 has an internal stopcodon in comparison with FCTR3. See Table 8. Unlike FCTR3, however,FCTR4 lacks a significant portion of the PDGF-like domain. See Table 9.

TABLE 5 Nucleotide (SEQ ID NO:9) and Protein (SEQ ID NO:10) Sequence ofFCTR4 ATGCAACGGCTCGTTTTAGTCTCCATTCTCCTGTGCGCGAACTTTAGCTGCTATCCGGACACTTTTGCGACTCCGCAGAGAGCATCCATCAAAGCTTTGCGCAATGCCAACCTCAGGAGAGATGAGAGCAATCACCTCACAGACTTGTACCAGAGAGAGGAGAACATTCAGGTGACAAGCAATGGCCATGTGCAGAGTCCTCGCTTCCCGAACAGCTACCCAAGGAACCTGCTTCTGACATGGTGGCTCCGTTCCCAGGAGAAAACACGGATACAACTGTCCTTTGACCATCAATTCGGACTAGAGGAAGCAGAAAATGACATTTGTAGGTATGACTTTGTGGAAGTTGAAGAAGTCTCAGAGAGCAGCACTGTTGTCAGAGGAAGATGGTGTGGCCACAAGGAGATCCCTCCAAGGATAACGTCAAGAACAAACCAGATTAAAATCACATTTAAGTCTGATGACTACTTTGTGGCAAAACCTGGATTCAAGATTTATTATTCATTTGTGGAAGATTTCCAACCGGAAGCAGCCTCAGAGACCAACTGGGAATCAGTCACAAGCTCTTTCTCTGGGGTGTCCTATCACTCTCCATCAATAACGGACCCCACTCTCACTGCTGATGCCCTGGACAAAACTGTCGCAGAATTCGATACCGTGGAAGATCTACTTAAGCACTTCAATCCAGTGTCTTGGCAAGATGATCTGGAGAATTTGTATCTGGACACCCCTCATTATAGAGGCAGGTCATACCATGATCGGAAGTCCAAAGGTATTGAAGTTTGAGCCTGGACATTTCAAGAGAAGGGGCAAAGCTAAGAATATGGCTCTTGTTGATATCCAGCTGGATCATCATGAGCGATGTGACTGTATCTGCAGCTCAAGACCACCTCGATAAMQRLVLVSILLCANFSCYPDTFATPQRASIKALRNANLRRDESNHLTDLYQREENIQVTSNGHVQSPRFPNSYPRNLLLTWWLRSQEKTRIQLSFDHQFGLEEAENDTCRYDFVEVEEVSESSTVVRGRWCGHKEIPPRITSRTNQIKITFKSDDYFVAKPGFKIYYSFVEDFQPEAASETNWESVTSSFSGVSYHSPSITDPTLTADALDKTVAEFDTVEDLLKHFNPVSWQDDLENLYLDTPHYRGRS YHDRKSKGIEVFCTR5 Nucleic Acids and Polypeptides

A FCTR5 (also referred to within the specification as PDGFD or humanPDGFD or hPDGFD or clone pCR2.1-S852_(—)2B) nucleic acid and polypeptideaccording to the invention includes the nucleic acid and encodedpolypeptide sequence of FCTR5 and is shown in Table 6 (SEQ ID NO:11 andSEQ ID NO:12). The FCTR5 nucleic acid sequence was identified as asplice variant of FCTR1.

Similar to FCTR1, protein structure analysis programs PSORT, PFAM andPROSITE predicted that FCTR5 contains a characteristic signal peptide(aa 1-23), PDGF domain (aa 272-362) and a N-linked glycosylation site(residue 276). BLASTP analysis revealed that the human FGTR5 is mostclosely related to human PDGF C, PDGF B, and PDGF A (42%, 27%, and 25%overall amino acid identity, respectively).

TABLE 6 Nucleotide (SEQ ID NO:11) and Protein (SEQ ID NO:12) Sequence ofFCTR5 ATGCACCGGCTCATCTTGTTCTACACTCTAATCTGCGCAAACTTTTGCAGCTGTCGGGACACTTCTGCAACCCCGCAGAGCGCATCCATCAAAGCTTTGCGCAACGCCAACCTCAGGCGAGATGTTGACCTGGATAGGCTCAATGATGATGCCAAGCGTTACAGTTGCACTCCCAGGAATTACTCGGTCAATATAAGAGAAGAGCTGAAGTTGGCCAATGTGGTCTTCTTTCCACGTTGCCTCCTCGTGCAGCCCTGTGGAGGAAATTGTGGCTGTGGAACTGTCAACTGGAGGTCCTGCACATGCAATTCAGGGAAAACCGTGAAAAAGTATCATGAGGTATTACAGTTTGAGCCTGGCCACATCAAGAGGAGGGGTAGAGCTAAGACCATGGCTCTAGTTGACATCCAGTTGGATCACCATGAACGATGCGATTGTATCTGCAGCTCA AGACCACCTCGAMHRLILFYTLICANFCSCRDTSATPQSASIKALRNANLRRDVDLDRLNDDAKRYSCTPRNYSVNIREELKLANVVFFPRCLLVQRCGGNCGCGTVNWRSCTCNSGKTVKKYHEVLQFEPGHIKRRGRAKTMALVDIQLDHHERCDCICSS RPPRFCTR6 Nucleic Acids and Polypeptides

A FCTR6 (also referred to within the specification as PDGFD or humanPDGFD or hPDGFD) nucleic acid and polypeptide according to the inventionincludes the nucleic acid and encoded polypeptide sequence of FCTR6 andis shown in Table 7 (SEQ ID NO:13 and SEQ ID NO:14). The FCTR6 sequence(also referred to as clone pCR2.1-S869_(—)4B) was identified as a splicevariant of FCTR1.

FCTR6 contains much of the 5′ end of the full length gene (FCTR1), butit is spliced to a cryptic, non-consensus splice site at the extreme 3′end of the coding sequence. This splicing introduces a STOP codonimmediately downstream to the splice site. This splice variant containsthe intact CUB domain of 30664188.0.99, but deletes the PDGF domains,indicating a possible regulatory function of the molecule.

Similar to FCTR1, however, protein structure analysis programs PSORT,PFAM and PROSITE predicted that FCTR6 contains a characteristic signalpeptide (aa 1-23), a CUB domain (aa 53-167) and an N-linkedglycosylation site (residue 276). BLASTP analysis revealed that thehuman FGTR5 is most closely related to human PDGF C, PDGF B, and PDGF A(42%, 27%, and 25% overall amino acid identity, respectively).

TABLE 7 Nucleotide (SEQ ID NO:13) and Protein (SEQ ID NO:14) Sequence ofFCTR6 ATGCACCGGCTCATCTTTGTCTACACTCTAATCTGCGCAAACTTTTGCAGCTGTCGGGACACTTCTGCAACCCCGCAGAGCGCATCCATCAAAGCTTTGCGCAACGCCAACCTCAGGCGAGATGAGAGCAATCACCTCACAGACTTGTACCGAAGAGATGAGACCATCCAGGTGAAGGAAACGGCTACGTGCAGAGTCCTAGATTCCCGAACAGCTACCCCAGGAACCTGCTCCTGACATGGCGGCTTCACTCTCAGGAGAATACACGGATACAGCTAGTGTTTGACAATCAGTTTGGATTAGAGGAAGCAGAAAATGATATCTGTAGGTAGAGCTAAGACCATGGCTCTAGTTGACATCCAGTTGGATCACCATGAACGATGCGATTGTATCTGCAGCT CAAGACCACCTCGAMHRLIFVYTLICANFCSCRDTSATPQSASIKALRNANLRRDESNHLTDLYRRDETIQVKGNGYVQSPRFPNSYPRNLLLTWRLHSQENTRIQLVFDNQFG LEEAENDICRFCTRX Sequences

The various FCTRX nucleic acids and polypeptides are disclosed inrelated applications U.S. Ser. No. 60/158,083, filed Oct. 7, 1999; U.S.Ser. No. 60/159,231, filed Oct. 13, 1999; U.S. Ser. No. 60/174,485 filedJan. 4, 2000; U.S. Ser. No. 60/186,707 filed Mar. 3, 2000; U.S. Ser. No.60/188,250, filed Mar. 10, 2000; U.S. Ser. No. 60/223,879, filed Aug. 8,2000; U.S. Ser. No. 60/234,082, filed on Sep. 20, 2000; U.S. Ser. No.09/685,330, filed on Oct. 5, 2000; PCT Application US00/27671, filedOct. 6, 2000; U.S. Ser. No. 09/688,312, filed Oct. 13, 2000; U.S. Ser.No. 09/715,332 filed Nov. 16, 2000; and U.S. Ser. No. 09/775,482 filedFeb. 2, 2001. Each of these applications is incorporated by reference inits entirety.

FCTRX amino acid sequence variants were analyzed with ClustalW software.The resulting sequence alignment is shown in Table 8.

TABLE 8 Alignment of FCTRX Polypeptide Sequences.

Nucleic acids of FCTR1, FCTR3, FCTR4 and FCTR6 are aligned with eachother over the nucleotide residues shown in Table 9.

TABLE 9 Alignment of SEQ ID NOS:3, 7, 9 and 13, respectively.

Amino acids of FCTR1, FCTR3, FCTR4 and FCTR6 are aligned with each otheras shown in Table 10.

TABLE 10 Alignment of SEQ ID NOS:4, 8, 10 and 14, respectively.

Nucleic acids of FCTR2 and FCTR5 are aligned with each other over thenucleotide residues shown in Table 11.

TABLE 11 Alignment of SEQ ID NO:5 and SEQ ID NO:11, respectively.

Amino acids of FCTR2 and FCTR5 are aligned with each other as shown inTable 12.

TABLE 12 Alignment of SEQ ID NO:6 and SEQ ID NO:12, respectively.

The similarities of the disclosed FCTRX polypeptides to previouslydescribed BMP-1 VEGF-E and PDGF polypeptides indicate a similarity offunctions by the FCTRX nucleic acids and polypeptides of the invention.These utilities are described in more detail below.

FCTRX nucleic acids and polypeptides may be use to induce formation ofcartilage, as BMP-1 is also capable of inducing formation of cartilagein vivo (Wozney et al., Science 242: 1528-1534 (1988)).

An additional use for the FCTRX nucleic acids and polypeptides is in themodulation of collagen formation. Recombinantly expressed BMP1 andpurified procollagen C proteinase (PCP), a secreted metalloproteaserequiring calcium and needed for cartilage and bone formation, are, infact, identical. See, Kessler et al., Science 271:360-62 (1996). BMP-1cleaves the C-terminal propeptides of procollagen I, II, and III and itsactivity is increased by the procollagen C-endopeptidase enhancerprotein. FCTRX nucleic acids and polypeptides may play similar roles incollagen modulation pathways.

It is shown in the Examples below that FCTRX polypeptides have theability to reduce or ameliorate the extent of inflammatory response intwo animal models of inflammatory bowel disease.

The similarity between FCTRX polypeptides and PDGF polypeptides suggeststhat FCTRX nucleic acids and their encoded polypeptides can be used invarious therapeutic and diagnostic applications. For example, FCTRXnucleic acids and their encoded polypeptides can be used to treatcancer, cardiovascular and fibrotic diseases and diabetic ulcers. Inaddition, FCTRX nucleic acids and their encoded polypeptides will betherapeutically useful for the prevention of aneurysms and the theacceleration of wound closure through gene therapy. Furthermore, FCTRXnucleic acids and their encoded polypeptides can be utilized tostimulate cellular growth.

A FCTRX nucleic acid or gene product, e.g., a nucleic acid encoding SEQID NO:4 or SEQ ID NO:6, is useful as a therapeutic agent in promotingwound healing, neovascularization and tissue growth, and similar tissueregeneration needs. More specifically, a FCTRX nucleic acid orpolypeptide may be useful in treatment of anemia and leukopenia,intestinal tract sensitivity and baldness. Treatment of such conditionsmay be indicated in, e.g., patients having undergone radiation orchemotherapy. It is intended in such cases that administration of a FCTXnucleic acid or polypeptide, e.g., a polypeptide including the aminoacid sequence of SEQ ID NO:4 or SEQ ID NO:6, or a nucleic acid sequenceencoding these polypeptides (e.g., SEQ ID NO:3 or SEQ ID NO:5) will becontrolled in dose such that any hyperproliferative side effects areminimized.

The invention also includes mature FGF-CX and/or FCTRX polypeptides,variants of mature FGF-CX and/or FCTRX polypeptides, fragments of matureand mature variant FGF-CX and/or FCTRX polypeptides, and nucleic acidsencoding these polypeptides and fragments. As used herein, a “mature”form of a FGF-CX and/or FCTRX polypeptide or protein disclosed in thepresent invention is the product of a naturally occurring polypeptide orprecursor form or proprotein. The naturally occurring polypeptide,precursor or proprotein includes, by way of nonlimiting example, thefull length gene product, encoded by the corresponding gene. In someembodiments, the mature form include an FGF-CX and/or FCTRX polypeptide,precursor or proprotein encoded by an open reading frame describedherein. The product “mature” form can arise, e.g., as a result of one ormore naturally occurring processing steps as they may take place withinthe cell, or host cell, in which the gene product arises.

Examples of such processing steps leading to a “mature” form of apolypeptide or protein include the cleavage of the N-terminal methionineresidue encoded by the initiation codon of an open reading frame, or theproteolytic cleavage of a signal peptide or leader sequence. Thus amature form arising from a FGF-CX or a FCTRX precursor polypeptide orprotein that has residues 1 to N, where residue 1 is the N-terminalmethionine, would have residues 2 through N remaining after removal ofthe N-terminal methionine. Alternatively, a mature form arising from aprecursor polypeptide or protein having residues 1 to N, in which anN-terminal signal sequence from residue 1 to residue M is cleaved, wouldhave the residues from residue M+1 to residue N remaining. Additionally,a “mature” protein or fragment may arise from a cleavage event otherthan removal of an initiating methionine or removal of a signal peptide.Further as used herein, a “mature” form of a FGF-CX and/or a FCTRXpolypeptide or protein may arise from a step of post-translationalmodification other than a proteolytic cleavage event. Such additionalprocesses include, by way of non-limiting example, glycosylation,myristoylation or phosphorylation. In general, a mature polypeptide orprotein may result from the operation of only one of these processes, ora combination of any of them.

As used herein, “identical” residues correspond to those residues in acomparison between two sequences where the equivalent nucleotide base oramino acid residue in an alignment of two sequences is the same residue.Residues are alternatively described as “similar” or “positive” when thecomparisons between two sequences in an alignment show that residues inan equivalent position in a comparison are either the same amino acid ora conserved amino acid as defined below.

Included within the invention are FGF-CX and FCTRX nucleic acids,isolated nucleic acids that encode FGF-CX and FCTRX polypeptides or aportion thereof, FGF-CX and FCTRX polypeptides, vectors containing thesenucleic acids, host cells transformed with the FGF-CX and/or FCTRXnucleic acids, anti-FGF-CX and/or FCTRX antibodies, and pharmaceuticalcompositions. Also disclosed are methods of making FGF-CX and/or FCTRXpolypeptides, as well as methods of screening, diagnosing, treatingconditions using these compounds, and methods of screening compoundsthat modulate FGF-CX and/or FCTRX polypeptide activity. The FGF-CXand/or FCTRX nucleic acids and polypeptides, as well as FGF-CX and/orFCTRX antibodies, therapeutic agents and pharmaceutical compositionsdiscussed herein, are useful, inter alia, in treating inflammatoryconditions, as well as tissue proliferation-associated disorders.

FGF-CX and/or FCTRX Nucleic Acids and Polypeptides

A summary of the FGF-CX and/or FCTRX nucleic acids and proteins of theinvention is provided in Table 13.

TABLE 13 Summary Of Nucleic Acids And Proteins Of The Invention NucleicAmino Acid SEQ Acid SEQ Clone Table Clone alias ID NO ID NO FGF-CX 1AB020858; CG53135-01; 1 2 CG53135-02; TA-AB02085- S274-F19; 20858 FCTR12 PDGFD; 30664188; 3 4 30664188.0.99; CG52053; CG52053-02;30664188.0.m99; 30664188-S311a; 30664188-S11a FCTR2 3 PDGFD;30664188.0.331; 5 6 CG52053-01 FCTR3 4 PDGFD; murine PDGFD; 7 8 mPDGFDFCTR4 5 PDGFD; murine PDGFD; 9 10 mPDGFD FCTR5 6 PDGFD; human PDGFD; 1112 hPDGFD; clone pCR2.1-S852_2B FCTR6 7 PDGFD; human PDGFD; 13 14hPDGFD; clone pCR2.1-S869_4B

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode FGF-CX and/or FCTRX polypeptides or biologically activeportions thereof. Also included in the invention are nucleic acidfragments sufficient for use as hybridization probes to identify FGF-CXand/or FCTRX-encoding nucleic acids (e.g., FGF-CX and/or FCTRX mRNAs)and fragments for use as PCR primers for the amplification and/ormutation of FGF-CX and/or FCTRX nucleic acid molecules. As used herein,the term “nucleic acid molecule” is intended to include DNA molecules(e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of theDNA or RNA generated using nucleotide analogs, and derivatives,fragments and homologs thereof. The nucleic acid molecule may besingle-stranded or double-stranded, but preferably is compriseddouble-stranded DNA.

An FGF-CX and/or FCTRX nucleic acid can encode a mature FGF-CX and/orFCTRX polypeptide. As used herein, a “mature” form of a polypeptide orprotein disclosed in the present invention is the product of a naturallyoccurring polypeptide or precursor form or proprotein. The naturallyoccurring polypeptide, precursor or proprotein includes, by way ofnonlimiting example, the full-length gene product, encoded by thecorresponding gene. Alternatively, it may be defined as the polypeptide,precursor or proprotein encoded by an ORF described herein. The product“mature” form arises, again by way of nonlimiting example, as a resultof one or more naturally occurring processing steps as they may takeplace within the cell, or host cell, in which the gene product arises.Examples of such processing steps leading to a “mature” form of apolypeptide or protein include the cleavage of the N-terminal methionineresidue encoded by the initiation codon of an ORF, or the proteolyticcleavage of a signal peptide or leader sequence. Thus a mature formarising from a precursor polypeptide or protein that has residues 1 toN, where residue 1 is the N-terminal methionine, would have residues 2through N remaining after removal of the N-terminal methionine.Alternatively, a mature form arising from a precursor polypeptide orprotein having residues 1 to N, in which an N-terminal signal sequencefrom residue 1 to residue M is cleaved, would have the residues fromresidue M+1 to residue N remaining. Further as used herein, a “mature”form of a polypeptide or protein may arise from a step ofpost-translational modification other than a proteolytic cleavage event.Such additional processes include, by way of non-limiting example,glycosylation, myristoylation or phosphorylation. In general, a maturepolypeptide or protein may result from the operation of only one ofthese processes, or a combination of any of them.

The term “probes”, as utilized herein, refers to nucleic acid sequencesof variable length, preferably between at least about 10 nucleotides(nt), 100 nt, or as many as approximately, e.g., 6,000 nt, dependingupon the specific use. Probes are used in the detection of identical,similar, or complementary nucleic acid sequences. Longer length probesare generally obtained from a natural or recombinant source, are highlyspecific, and much slower to hybridize than shorter-length oligomerprobes. Probes may be single- or double-stranded and designed to havespecificity in PCR, membrane-based hybridization technologies, orELISA-like technologies.

The term “isolated” nucleic acid molecule, as utilized herein, is one,which is separated from other nucleic acid molecules which are presentin the natural source of the nucleic acid. Preferably, an “isolated”nucleic acid is free of sequences which naturally flank the nucleic acid(i.e., sequences located at the 5′- and 3′-termini of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated FGF-CX and/orFCTRX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturallyflank the nucleic acid molecule in genomic DNA of the cell/tissue fromwhich the nucleic acid is derived (e.g., brain, heart, liver, spleen,etc.). Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material orculture medium when produced by recombinant techniques, or of chemicalprecursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the invention, e.g., a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13,or a complement of this aforementioned nucleotide sequence, can beisolated using standard molecular biology techniques and the sequenceinformation provided herein. Using all or a portion of the nucleic acidsequence of SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13 as a hybridizationprobe, FGF-CX and/or FCTRX molecules can be isolated using standardhybridization and cloning techniques (e.g., as described in Sambrook, etal., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2^(nd) Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; andAusubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, JohnWiley & Sons, New York, N.Y., 1993.)

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to FGF-CX and/or FCTRXnucleotide sequences can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

As used herein, the term “oligonucleotide” refers to a series of linkednucleotide residues, which oligonucleotide has a sufficient number ofnucleotide bases to be used in a PCR reaction. A short oligonucleotidesequence may be based on, or designed from, a genomic or cDNA sequenceand is used to amplify, confirm, or reveal the presence of an identical,similar or complementary DNA or RNA in a particular cell or tissue.Oligonucleotides comprise portions of a nucleic acid sequence havingabout 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 ntin length. In one embodiment of the invention, an oligonucleotidecomprising a nucleic acid molecule less than 100 nt in length wouldfurther comprise at least 6 contiguous nucleotides of SEQ ID NOS:1, 3,5, 7, 9, 11 and 13, or a complement thereof. Oligonucleotides may bechemically synthesized and may also be used as probes.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule that is a complement of thenucleotide sequence shown in SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13, or aportion of this nucleotide sequence (e.g., a fragment that can be usedas a probe or primer or a fragment encoding a biologically-activeportion of an FGF-CX and/or FCTRX polypeptide). A nucleic acid moleculethat is complementary to the nucleotide sequence shown in SEQ ID NOS:1,3, 5, 7, 9, 11 and 13 is one that is sufficiently complementary to thenucleotide sequence shown in SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13 that itcan hydrogen bond with little or no mismatches to the nucleotidesequence shown SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13, thereby forming astable duplex.

As used herein, the term “complementary” refers to Watson-Crick orHoogsteen base pairing between nucleotides units of a nucleic acidmolecule, and the term “binding” means the physical or chemicalinteraction between two polypeptides or compounds or associatedpolypeptides or compounds or combinations thereof. Binding includesionic, non-ionic, van der Waals, hydrophobic interactions, and the like.A physical interaction can be either direct or indirect. Indirectinteractions may be through or due to the effects of another polypeptideor compound. Direct binding refers to interactions that do not takeplace through, or due to, the effect of another polypeptide or compound,but instead are without other substantial chemical intermediates.

Fragments provided herein are defined as sequences of at least 6(contiguous) nucleic acids or at least 4 (contiguous) amino acids, alength sufficient to allow for specific hybridization in the case ofnucleic acids or for specific recognition of an epitope in the case ofamino acids, respectively, and are at most some portion less than a fulllength sequence. Fragments may be derived from any contiguous portion ofa nucleic acid or amino acid sequence of choice. Derivatives are nucleicacid sequences or amino acid sequences formed from the native compoundseither directly or by modification or partial substitution. Analogs arenucleic acid sequences or amino acid sequences that have a structuresimilar to, but not identical to, the native compound but differs fromit in respect to certain components or side chains. Analogs may besynthetic or from a different evolutionary origin and may have a similaror opposite metabolic activity compared to wild type. Homologs arenucleic acid sequences or amino acid sequences of a particular gene thatare derived from different species.

Derivatives and analogs may be full length or other than full length, ifthe derivative or analog contains a modified nucleic acid or amino acid,as described below. Derivatives or analogs of the nucleic acids orproteins of the invention include, but are not limited to, moleculescomprising regions that are substantially homologous to the nucleicacids or proteins of the invention, in various embodiments, by at leastabout 70%, 80%, or 95% identity (with a preferred identity of 80-95%)over a nucleic acid or amino acid sequence of identical size or whencompared to an aligned sequence in which the alignment is done by acomputer homology program known in the art, or whose encoding nucleicacid is capable of hybridizing to the complement of a sequence encodingthe aforementioned proteins under stringent, moderately stringent, orlow stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993, and below.

A “homologous nucleic acid sequence” or “homologous amino acidsequence,” or variations thereof, refer to sequences characterized by ahomology at the nucleotide level or amino acid level as discussed above.Homologous nucleotide sequences encode those sequences coding forisoforms of FGF-CX and/or FCTRX polypeptides. Isoforms can be expressedin different tissues of the same organism as a result of, for example,alternative splicing of RNA. Alternatively, isoforms can be encoded bydifferent genes. In the invention, homologous nucleotide sequencesinclude nucleotide sequences encoding for an FGF-CX and/or FCTRXpolypeptide of species other than humans, including, but not limited to:vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog,cat cow, horse, and other organisms. Homologous nucleotide sequencesalso include, but are not limited to, naturally occurring allelicvariations and mutations of the nucleotide sequences set forth herein. Ahomologous nucleotide sequence does not, however, include the exactnucleotide sequence encoding human FGF-CX and/or FCTRX protein.Homologous nucleic acid sequences include those nucleic acid sequencesthat encode conservative amino acid substitutions (see below) in SEQ IDNOS:1, 3, 5, 7, 9, 11 and 13, as well as a polypeptide possessing FGF-CXand/or FCTRX biological activity. Various biological activities of theFGF-CX and/or FCTRX proteins are described below.

As used herein, “identical” residues correspond to those residues in acomparison between two sequences where the equivalent nucleotide base oramino acid residue in an alignment of two sequences is the same residue.Residues are alternatively described as “similar” or “positive” when thecomparisons between two sequences in an alignment show that residues inan equivalent position in a comparison are either the same amino acid ora conserved amino acid as defined below.

An FGF-CX and/or FCTRX polypeptide is encoded by the open reading frame(“ORF”) of an FGF-CX and/or FCTRX nucleic acid. An ORF corresponds to anucleotide sequence that could potentially be translated into apolypeptide. A stretch of nucleic acids comprising an ORF isuninterrupted by a stop codon. An ORF that represents the codingsequence for a full protein begins with an ATG “start” codon andterminates with one of the three “stop” codons, namely, TAA, TAG, orTGA. For the purposes of this invention, an ORF may be any part of acoding sequence, with or without a start codon, a stop codon, or both.For an ORF to be considered as a good candidate for coding for abonafide cellular protein, a minimum size requirement is often set,e.g., a stretch of DNA that would encode a protein of 50 amino acids ormore.

The nucleotide sequences determined from the cloning of the human FGF-CXand/or FCTRX genes allows for the generation of probes and primersdesigned for use in identifying and/or cloning FGF-CX and/or FCTRXhomologues in other cell types, e.g. from other tissues, as well asFGF-CX and/or FCTRX homologues from other vertebrates. The probe/primertypically comprises substantially purified oligonucleotide. Theoligonucleotide typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12, 25, 50, 100,150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotidesequence of SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13; or an anti-sense strandnucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13; or of anaturally occurring mutant of SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13.

Probes based on the human FGF-CX and/or FCTRX nucleotide sequences canbe used to detect transcripts or genomic sequences encoding the same orhomologous proteins. In various embodiments, the probe further comprisesa label group attached thereto, e.g. the label group can be aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as a part of a diagnostic test kit foridentifying cells or tissues which mis-express an FGF-CX and/or FCTRXprotein, such as by measuring a level of an FGF-CX and/or FCTRX-encodingnucleic acid in a sample of cells from a subject e.g., detecting FGF-CXand/or FCTRX mRNA levels or determining whether a genomic FGF-CX and/orFCTRX gene has been mutated or deleted.

“A polypeptide having a biologically-active portion of an FGF-CX and/orFCTRX polypeptide” refers to polypeptides exhibiting activity similar,but not necessarily identical to, an activity of a polypeptide of theinvention, including mature forms, as measured in a particularbiological assay, with or without dose dependency. A nucleic acidfragment encoding a “biologically-active portion of FGF-CX and/or FCTRX”can be prepared by isolating a portion SEQ ID NOS:1, 3, 5, 7, 9, 11 and13 that encodes a polypeptide having an FGF-CX and/or FCTRX biologicalactivity (the biological activities of the FGF-CX and/or FCTRX proteinsare described below), expressing the encoded portion of FGF-CX and/orFCTRX protein (e.g., by recombinant expression in vitro) and assessingthe activity of the encoded portion of FGF-CX and/or FCTRX.

FGF-CX and/or FCTRX Nucleic Acid and Polypeptide Variants

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequences shown SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13due to degeneracy of the genetic code and thus encode the same FGF-CXand/or FCTRX proteins as that encoded by the nucleotide sequences shownin SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13. In another embodiment, anisolated nucleic acid molecule of the invention has a nucleotidesequence encoding a protein having an amino acid sequence shown in SEQID NOS:2, 4, 6, 8, 10, 12 and 14.

In addition to the human FGF-CX and/or FCTRX nucleotide sequences shownin SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13 it will be appreciated by thoseskilled in the art that DNA sequence polymorphisms that lead to changesin the amino acid sequences of the FGF-CX and/or FCTRX polypeptides mayexist within a population (e.g., the human population). Such geneticpolymorphism in the FGF-CX and/or FCTRX genes may exist amongindividuals within a population due to natural allelic variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame (ORF) encoding an FGF-CXand/or FCTRX protein, preferably a vertebrate FGF-CX and/or FCTRXprotein. Such natural allelic variations can typically result in 1-5%variance in the nucleotide sequence of the FGF-CX and/or FCTRX genes.Any and all such nucleotide variations and resulting amino acidpolymorphisms in the FGF-CX and/or FCTRX polypeptides, which are theresult of natural allelic variation and that do not alter the functionalactivity of the FGF-CX and/or FCTRX polypeptides, are intended to bewithin the scope of the invention.

Moreover, nucleic acid molecules encoding FGF-CX and/or FCTRX proteinsfrom other species, and thus that have a nucleotide sequence thatdiffers from the human sequence SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13 areintended to be within the scope of the invention. Nucleic acid moleculescorresponding to natural allelic variants and homologues of the FGF-CXand/or FCTRX cDNAs of the invention can be isolated based on theirhomology to the human FGF-CX and/or FCTRX nucleic acids disclosed hereinusing the human cDNAs, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13. In anotherembodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750,1000, 1500, or 2000 or more nucleotides in length. In yet anotherembodiment, an isolated nucleic acid molecule of the inventionhybridizes to the coding region. As used herein, the term “hybridizesunder stringent conditions” is intended to describe conditions forhybridization and washing under which nucleotide sequences at least 60%homologous to each other typically remain hybridized to each other.

Homologs (i.e., nucleic acids encoding FGF-CX and/or FCTRX proteinsderived from species other than human) or other related sequences (e.g.,paralogs) can be obtained by low, moderate or high stringencyhybridization with all or a portion of the particular human sequence asa probe using methods well known in the art for nucleic acidhybridization and cloning.

As used herein, the phrase “stringent hybridization conditions” refersto conditions under which a probe, primer or oligonucleotide willhybridize to its target sequence, but to no other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures than shorter sequences. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Since thetarget sequences are generally present at excess, at Tm, 50% of theprobes are occupied at equilibrium. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes,primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about60° C. for longer probes, primers and oligonucleotides. Stringentconditions may also be achieved with the addition of destabilizingagents, such as formamide.

Stringent conditions are known to those skilled in the art and can befound in Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, theconditions are such that sequences at least about 65%, 70%, 75%, 85%,90%, 95%, 98%, or 99% homologous to each other typically remainhybridized to each other. A non-limiting example of stringenthybridization conditions are hybridization in a high salt buffercomprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02%PVP; 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNAat 65° C., followed by one or more washes in 0.2×SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizesunder stringent conditions to the sequences of SEQ ID NOS:1, 3, 5, 7, 9,11 and 13 corresponds to a naturally-occurring nucleic acid molecule. Asused herein, a “naturally-occurring” nucleic acid molecule refers to anRNA or DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein).

In a second embodiment, a nucleic acid sequence that is hybridizable tothe nucleic acid molecule comprising the nucleotide sequence of SEQ IDNOS:1, 3, 5, 7, 9, 11 and 13 or fragments, analogs or derivativesthereof, under conditions of moderate stringency is provided. Anon-limiting example of moderate stringency hybridization conditions arehybridization in 6×SSC, 5× Denhardt's solution, 0.5% SDS and 100 mg/mldenatured salmon sperm DNA at 55° C., followed by one or more washes in1×SSC, 0.1% SDS at 37° C. Other conditions of moderate stringency thatmay be used are well-known within the art. See, e.g., Ausubel, et al.(eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons,NY, and Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORYMANUAL, Stockton Press, NY.

In a third embodiment, a nucleic acid that is hybridizable to thenucleic acid molecule comprising the nucleotide sequences of SEQ IDNOS:1, 3, 5, 7, 9, 11 and 13 or fragments, analogs or derivativesthereof, under conditions of low stringency, is provided. A non-limitingexample of low stringency hybridization conditions are hybridization in35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP,0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%(wt/vol) dextran sulfate at 40° C., followed by one or more washes in2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50° C. Otherconditions of low stringency that may be used are well known in the art(e.g., as employed for cross-species hybridizations). See, e.g.,Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION,A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. ProcNatl Acad Sci USA 78: 6789-6792.

Conservative Mutations

In addition to naturally-occurring allelic variants of FGF-CX and/orFCTRX sequences that may exist in the population, the skilled artisanwill further appreciate that changes can be introduced by mutation intothe nucleotide sequences of SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13 therebyleading to changes in the amino acid sequences of the encoded FGF-CXand/or FCTRX proteins, without altering the functional ability of saidFGF-CX and/or FCTRX proteins. For, example, nucleotide substitutionsleading to amino acid substitutions at “non-essential” amino acidresidues can be made in the sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12and 14. A “non-essential” amino acid residue is a residue that can bealtered from the wild-type sequences of the FGF-CX and/or FCTRX proteinswithout altering their biological activity, whereas an “essential” aminoacid residue is required for such biological activity. For example,amino acid residues that are conserved among the FGF-CX and/or FCTRXproteins of the invention are predicted to be particularly non-amenableto alteration. Amino acids for which conservative substitutions can bemade are well-known within the art.

Another aspect of the invention pertains to nucleic acid moleculesencoding FGF-CX and/or FCTRX proteins that contain changes in amino acidresidues that are not essential for activity. Such FGF-CX and/or FCTRXproteins differ in amino acid sequence from SEQ ID NOS:2, 4, 6, 8, 10,12 and 14 yet retain biological activity. In one embodiment, theisolated nucleic acid molecule comprises a nucleotide sequence encodinga protein, wherein the protein comprises an amino acid sequence at leastabout 45% homologous to the amino acid sequences of SEQ ID NOS:2, 4, 6,8, 10, 12 and 14. Preferably, the protein encoded by the nucleic acidmolecule is at least about 60% homologous to SEQ ID NOS:2, 4, 6, 8, 10,12 and 14; more preferably at least about 70% homologous to SEQ IDNOS:2, 4, 6, 8, 10, 12 and 14; still more preferably at least about 80%homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12 and 14; even more preferablyat least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12 and 14;and most preferably at least about 95% homologous to SEQ ID NOS:2, 4, 6,8, 10, 12 and 14.

An isolated nucleic acid molecule encoding an FGF-CX and/or FCTRXprotein homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12 and14 can be created by introducing one or more nucleotide substitutions,additions or deletions into the nucleotide sequence of SEQ ID NOS:1, 3,5, 7, 9, 11 and 13 such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.

Mutations can be introduced into SEQ ID NOS:2, 4, 6, 8, 10, 12 and 14 bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted, non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined within the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted non-essential amino acid residue in theFGF-CX and/or FCTRX protein is replaced with another amino acid residuefrom the same side chain family. Alternatively, in another embodiment,mutations can be introduced randomly along all or part of an FGF-CXand/or FCTRX coding sequence, such as by saturation mutagenesis, and theresultant mutants can be screened for FGF-CX and/or FCTRX biologicalactivity to identify mutants that retain activity. Following mutagenesisof SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13, the encoded protein can beexpressed by any recombinant technology known in the art and theactivity of the protein can be determined.

The relatedness of amino acid families may also be determined based onside chain interactions. Substituted amino acids may be fully conserved“strong” residues or fully conserved “weak” residues. The “strong” groupof conserved amino acid residues may be any one of the following groups:STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the singleletter amino acid codes are grouped by those amino acids that may besubstituted for each other. Likewise, the “weak” group of conservedresidues may be any one of the following: CSA, ATV, SAG, STNK, STPA,SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, HFY, wherein the letters within eachgroup represent the single letter amino acid code.

In one embodiment, a mutant FGF-CX and/or FCTRX protein can be assayedfor (i) the ability to form protein:protein interactions with otherFGF-CX and/or FCTRX proteins, other cell-surface proteins, orbiologically-active portions thereof, (ii) complex formation between amutant FGF-CX and/or FCTRX protein and an FGF-CX and/or FCTRX ligand; or(iii) the ability of a mutant FGF-CX and/or FCTRX protein to bind to anintracellular target protein or biologically-active portion thereof;(e.g. avidin proteins).

In yet another embodiment, a mutant FGF-CX and/or FCTRX protein can beassayed for the ability to regulate a specific biological function(e.g., regulation of insulin release).

Antisense Nucleic Acids

Another aspect of the invention pertains to isolated antisense nucleicacid molecules that are hybridizable to or complementary to the nucleicacid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 5,7, 9, 11 and 13, or fragments, analogs or derivatives thereof. An“antisense” nucleic acid comprises a nucleotide sequence that iscomplementary to a “sense” nucleic acid encoding a protein (e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence). In specific aspects, antisensenucleic acid molecules are provided that comprise a sequencecomplementary to at least about 10, 25, 50, 100, 250 or 500 nucleotidesor an entire FGF-CX and/or FCTRX coding strand, or to only a portionthereof. Nucleic acid molecules encoding fragments, homologs,derivatives and analogs of an FGF-CX and/or FCTRX protein of SEQ IDNOS:2, 4, 6, 8, 10, 12 and 14, or antisense nucleic acids complementaryto an FGF-CX and/or FCTRX nucleic acid sequence of SEQ ID NOS:1, 3, 5,7, 9, 11 and 13, are additionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence encodingan FGF-CX and/or FCTRX protein. The term “coding region” refers to theregion of the nucleotide sequence comprising codons which are translatedinto amino acid residues. In another embodiment, the antisense nucleicacid molecule is antisense to a “noncoding region” of the coding strandof a nucleotide sequence encoding the FGF-CX and/or FCTRX protein. Theterm “noncoding region” refers to 5′ and 3′ sequences which flank thecoding region that are not translated into amino acids (i.e., alsoreferred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding the FGF-CX and/or FCTRXprotein disclosed herein, antisense nucleic acids of the invention canbe designed according to the rules of Watson and Crick or Hoogsteen basepairing. The antisense nucleic acid molecule can be complementary to theentire coding region of FGF-CX and/or FCTRX mRNA, but more preferably isan oligonucleotide that is antisense to only a portion of the coding ornoncoding region of FGF-CX and/or FCTRX mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of FGF-CX and/or FCTRX mRNA. An antisenseoligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40, 45 or 50 nucleotides in length. An antisense nucleic acid of theinvention can be constructed using chemical synthesis or enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally-occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids (e.g,phosphorothioate derivatives and acridine substituted nucleotides can beused).

Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil, 5-methyluracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, uracil-5-oxyaceticacid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,2,6-diaminopurine, (acp3)w, and 3-(3-amino-3-N-2-carboxypropyl)uracil.Alternatively, the antisense nucleic acid can be produced biologicallyusing an expression vector into which a nucleic acid has been subclonedin an antisense orientation (i.e., RNA transcribed from the insertednucleic acid will be of an antisense orientation to a target nucleicacid of interest, described further in the following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding an FGF-CXand/or FCTRX protein to thereby inhibit expression of the protein (e.g.,by inhibiting transcription and/or translation). The hybridization canbe by conventional nucleotide complementarity to form a stable duplex,or, for example, in the case of an antisense nucleic acid molecule thatbinds to DNA duplexes, through specific interactions in the major grooveof the double helix. An example of a route of administration ofantisense nucleic acid molecules of the invention includes directinjection at a tissue site. Alternatively, antisense nucleic acidmolecules can be modified to target selected cells and then administeredsystemically. For example, for systemic administration, antisensemolecules can be modified such that they specifically bind to receptorsor antigens expressed on a selected cell surface (e.g., by linking theantisense nucleic acid molecules to peptides or antibodies that bind tocell surface receptors or antigens). The antisense nucleic acidmolecules can also be delivered to cells using the vectors describedherein. To achieve sufficient nucleic acid molecules, vector constructsin which the antisense nucleic acid molecule is placed under the controlof a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other. See, e.g., Gaultier, et al., 1987. Nucl. Acids Res. 15:6625-6641. The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (see, e.g., Inoue, et al. 1987. Nucl. AcidsRes. 15: 6131-6148) or a chimeric RNA-DNA analogue (see, e.g., Inoue, etal., 1987. FEBS Lett. 215: 327-330.

Ribozymes and PNA Moieties

Nucleic acid modifications include, by way of non-limiting example,modified bases, and nucleic acids whose sugar phosphate backbones aremodified or derivatized. These modifications are carried out at least inpart to enhance the chemical stability of the modified nucleic acid,such that they may be used, for example, as antisense binding nucleicacids in therapeutic applications in a subject.

In one embodiment, an antisense nucleic acid of the invention is aribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity that are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes as described in Haselhoff andGerlach 1988. Nature 334: 585-591) can be used to catalytically cleaveFGF-CX and/or FCTRX mRNA transcripts to thereby inhibit translation ofFGF-CX and/or FCTRX mRNA. A ribozyme having specificity for an FGF-CXand/or FCTRX-encoding nucleic acid can be designed based upon thenucleotide sequence of an FGF-CX and/or FCTRX cDNA disclosed herein(i.e., SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13). For example, a derivativeof a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in an FGF-CX and/or FCTRX-encoding mRNA. See, e.g., U.S.Pat. No. 4,987,071 to Cech, et al. and U.S. Pat. No. 5,116,742 to Cech,et al. FGF-CX and/or FCTRX mRNA can also be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.

Alternatively, FGF-CX and/or FCTRX gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofthe FGF-CX and/or FCTRX nucleic acid (e.g., the FGF-CX and/or FCTRXpromoter and/or enhancers) to form triple helical structures thatprevent transcription of the FGF-CX and/or FCTRX gene in target cells.See, e.g., Helene, 1991. Anticancer Drug Des. 6: 569-84; Helene, et al.1992. Ann. N.Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14:807-15.

In various embodiments, the FGF-CX and/or FCTRX nucleic acids can bemodified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids. See, e.g.,Hyrup, et al., 1996. Bioorg Med Chem 4: 5-23. As used herein, the terms“peptide nucleic acids” or “PNAs” refer to nucleic acid mimics (e.g.,DNA mimics) in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup, et al.,1996. supra; Perry-O'Keefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93:14670-14675.

PNAs of FGF-CX and/or FCTRX can be used in therapeutic and diagnosticapplications. For example, PNAs can be used as antisense or antigeneagents for sequence-specific modulation of gene expression by, e.g.,inducing transcription or translation arrest or inhibiting replication.PNAs of FGF-CX and/or FCTRX can also be used, for example, in theanalysis of single base pair mutations in a gene (e.g., PNA directed PCRclamping; as artificial restriction enzymes when used in combinationwith other enzymes, e.g., S₁ nucleases (see, Hyrup, et al., 1996.supra);or as probes or primers for DNA sequence and hybridization (see, Hyrup,et al., 1996, supra; Perry-O'Keefe, et al, 1996. supra).

In another embodiment, PNAs of FGF-CX and/or FCTRX can be modified,e.g., to enhance their stability or cellular uptake, by attachinglipophilic or other helper groups to PNA, by the formation of PNA-DNAchimeras, or by the use of liposomes or other techniques of drugdelivery known in the art. For example, PNA-DNA chimeras of FGF-CXand/or FCTRX can be generated that may combine the advantageousproperties of PNA and DNA. Such chimeras allow DNA recognition enzymes(e.g., RNase H and DNA polymerases) to interact with the DNA portionwhile the PNA portion would provide high binding affinity andspecificity. PNA-DNA chimeras can be linked using linkers of appropriatelengths selected in terms of base stacking, number of bonds between thenucleobases, and orientation (see, Hyrup, et al., 1996. supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup, etal., 1996. supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363.For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry, and modified nucleosideanalogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used between the PNA and the 5′ end of DNA. See,e.g., Mag, et al., 1989. Nucl Acid Res 17: 5973-5988. PNA monomers arethen coupled in a stepwise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment. See, e.g., Finn, et al., 1996.supra. Alternatively, chimeric molecules can be synthesized with a 5′DNA segment and a 3′ PNA segment. See, e.g., Petersen, et al., 1975.Bioorg. Med. Chem. Lett. 5: 1119-11124.

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger, et al., 1989. Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652;PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g.,PCT Publication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization triggered cleavage agents (see, e.g., Krol,et al., 1988. BioTechniques 6:958-976) or intercalating agents (see,e.g., Zon, 1988. Pharm. Res. 5: 539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,a hybridization triggered cross-linking agent, a transport agent, ahybridization-triggered cleavage agent, and the like.

FGF-CX and/or FCTRX Polypeptides

A polypeptide according to the invention includes a polypeptideincluding the amino acid sequence of FGF-CX and/or FCTRX polypeptideswhose sequences are provided in SEQ ID NOS:2, 4, 6, 8, 10, 12 and 14.The invention also includes a mutant or variant protein any of whoseresidues may be changed from the corresponding residues shown in SEQ IDNOS:2, 4, 6, 8, 10, 12 and 14 while still encoding a protein thatmaintains its FGF-CX and/or FCTRX activities and physiologicalfunctions, or a functional fragment thereof.

In general, an FGF-CX and/or FCTRX variant that preserves FGF-CX and/orFCTRX-like function includes any variant in which residues at aparticular position in the sequence have been substituted by other aminoacids, and further include the possibility of inserting an additionalresidue or residues between two residues of the parent protein as wellas the possibility of deleting one or more residues from the parentsequence. Any amino acid substitution, insertion, or deletion isencompassed by the invention. In favorable circumstances, thesubstitution is a conservative substitution as defined above.

One aspect of the invention pertains to isolated FGF-CX and/or FCTRXproteins, and biologically-active portions thereof, or derivatives,fragments, analogs or homologs thereof. Also provided are polypeptidefragments suitable for use as immunogens to raise anti-FGF-CX and/orFCTRX antibodies. In one embodiment, native FGF-CX and/or FCTRX proteinscan be isolated from cells or tissue sources by an appropriatepurification scheme using standard protein purification techniques. Inanother embodiment, FGF-CX and/or FCTRX proteins are produced byrecombinant DNA techniques. Alternative to recombinant expression, anFGF-CX and/or FCTRX protein or polypeptide can be synthesized chemicallyusing standard peptide synthesis techniques.

An “isolated” or “purified” polypeptide or protein orbiologically-active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourcefrom which the FGF-CX and/or FCTRX protein is derived, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized. The language “substantially free of cellular material”includes preparations of FGF-CX and/or FCTRX proteins in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly-produced. In one embodiment, the language“substantially free of cellular material” includes preparations ofFGF-CX and/or FCTRX proteins having less than about 30% (by dry weight)of non-FGF-CX and/or FCTRX proteins (also referred to herein as a“contaminating protein”), more preferably less than about 20% ofnon-FGF-CX and/or FCTRX proteins, still more preferably less than about10% of non-FGF-CX and/or FCTRX proteins, and most preferably less thanabout 5% of non-FGF-CX and/or FCTRX proteins. When the FGF-CX and/orFCTRX protein or biologically-active portion thereof isrecombinantly-produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the FGF-CX and/or FCTRX protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of FGF-CX and/or FCTRX proteins inwhich the protein is separated from chemical precursors or otherchemicals that are involved in the synthesis of the protein. In oneembodiment, the language “substantially free of chemical precursors orother chemicals” includes preparations of FGF-CX and/or FCTRX proteinshaving less than about 30% (by dry weight) of chemical precursors ornon-FGF-CX and/or FCTRX chemicals, more preferably less than about 20%chemical precursors or non-FGF-CX and/or FCTRX chemicals, still morepreferably less than about 10% chemical precursors or non-FGF-CX and/orFCTRX chemicals, and most preferably less than about 5% chemicalprecursors or non-FGF-CX and/or FCTRX chemicals.

Biologically-active portions of FGF-CX and/or FCTRX proteins includepeptides comprising amino acid sequences sufficiently homologous to orderived from the amino acid sequences of the FGF-CX and/or FCTRXproteins (e.g., the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8,10, 12 and 14) that include fewer amino acids than the full-lengthFGF-CX and/or FCTRX proteins, and exhibit at least one activity of anFGF-CX and/or FCTRX protein. Typically, biologically-active portionscomprise a domain or motif with at least one activity of the FGF-CXand/or FCTRX protein. A biologically-active portion of an FGF-CX and/orFCTRX protein can be a polypeptide which is, for example, 10, 25, 50,100 or more amino acid residues in length.

Moreover, other biologically-active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a nativeFGF-CX and/or FCTRX protein.

In an embodiment, the FGF-CX and/or FCTRX protein has an amino acidsequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12 and 14. In otherembodiments, the FGF-CX and/or FCTRX protein is substantially homologousto SEQ ID NOS:2, 4, 6, 8, 10, 12 and 14, and retains the functionalactivity of the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12 and 14, yetdiffers in amino acid sequence due to natural allelic variation ormutagenesis, as described in detail, below. Accordingly, in anotherembodiment, the FGF-CX and/or FCTRX protein is a protein that comprisesan amino acid sequence at least about 45% homologous to the amino acidsequence SEQ ID NOS:2, 4, 6, 8, 10, 12 and 14, and retains thefunctional activity of the FGF-CX and/or FCTRX proteins of SEQ ID NOS:2,4, 6, 8, 10, 12 and 14.

Determining Homology Between Two or More Sequences

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are homologous at that position(i.e., as used herein amino acid or nucleic acid “homology” isequivalent to amino acid or nucleic acid “identity”).

The nucleic acid sequence homology may be determined as the degree ofidentity between two sequences. The homology may be determined usingcomputer programs known in the art, such as GAP software provided in theGCG program package. See, Needleman and Wunsch, 1970. J Mol Biol 48:443-453. Using GCG GAP software with the following settings for nucleicacid sequence comparison: GAP creation penalty of 5.0 and GAP extensionpenalty of 0.3, the coding region of the analogous nucleic acidsequences referred to above exhibits a degree of identity preferably ofat least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS(encoding) part of the DNA sequence shown in SEQ ID NOS:1, 3, 5, 7, 9,11 and 13.

The term “sequence identity” refers to the degree to which twopolynucleotide or polypeptide sequences are identical on aresidue-by-residue basis over a particular region of comparison. Theterm “percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over that region of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I, in the case of nucleic acids) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the region ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. The term “substantialidentity” as used herein denotes a characteristic of a polynucleotidesequence, wherein the polynucleotide comprises a sequence that has atleast 80 percent sequence identity, preferably at least 85 percentidentity and often 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison region.

Chimeric and Fusion Proteins

The invention also provides FGF-CX and/or FCTRX chimeric or fusionproteins. As used herein, an FGF-CX and/or FCTRX “chimeric protein” or“fusion protein” comprises an FGF-CX and/or FCTRX polypeptideoperatively-linked to a non-FGF-CX and/or FCTRX polypeptide. An “FGF-CXand/or FCTRX polypeptide” refers to a polypeptide having an amino acidsequence corresponding to an FGF-CX and/or FCTRX protein (SEQ ID NOS:2,4, 6, 8, 10, 12 and 14), whereas a “non-FGF-CX and/or FCTRX polypeptide”refers to a polypeptide having an amino acid sequence corresponding to aprotein that is not substantially homologous to the FGF-CX and/or FCTRXprotein, e.g., a protein that is different from the FGF-CX and/or FCTRXprotein and that is derived from the same or a different organism.Within an FGF-CX and/or FCTRX fusion protein the FGF-CX and/or FCTRXpolypeptide can correspond to all or a portion of an FGF-CX and/or FCTRXprotein. In one embodiment, an FGF-CX and/or FCTRX fusion proteincomprises at least one biologically-active portion of an FGF-CX and/orFCTRX protein. In another embodiment, an FGF-CX and/or FCTRX fusionprotein comprises at least two biologically-active portions of an FGF-CXand/or FCTRX protein. In yet another embodiment, an FGF-CX and/or FCTRXfusion protein comprises at least three biologically-active portions ofan FGF-CX and/or FCTRX protein. Within the fusion protein, the term“operatively-linked” is intended to indicate that the FGF-CX and/orFCTRX polypeptide and the non-FGF-CX and/or FCTRX polypeptide are fusedin-frame with one another. The non-FGF-CX and/or FCTRX polypeptide canbe fused to the N-terminus or C-terminus of the FGF-CX and/or FCTRXpolypeptide.

In one embodiment, the fusion protein is a GST-FGF-CX and/or FCTRXfusion protein in which the FGF-CX and/or FCTRX sequences are fused tothe C-terminus of the GST (glutathione S-transferase) sequences. Suchfusion proteins can facilitate the purification of recombinant FGF-CXand/or FCTRX polypeptides.

In another embodiment, the fusion protein is an FGF-CX and/or FCTRXprotein containing a heterologous signal sequence at its N-terminus. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of FGF-CX and/or FCTRX can be increased through use of aheterologous signal sequence.

In yet another embodiment, the fusion protein is an FGF-CX and/orFCTRX-immunoglobulin fusion protein in which the FGF-CX and/or FCTRXsequences are fused to sequences derived from a member of theimmunoglobulin protein family. The FGF-CX and/or FCTRX-immunoglobulinfusion proteins of the invention can be incorporated into pharmaceuticalcompositions and administered to a subject to inhibit an interactionbetween an FGF-CX and/or FCTRX ligand and an FGF-CX and/or FCTRX proteinon the surface of a cell, to thereby suppress FGF-CX and/orFCTRX-mediated signal transduction in vivo. The FGF-CX and/orFCTRX-immunoglobulin fusion proteins can be used to affect thebioavailability of an FGF-CX and/or FCTRX cognate ligand. Inhibition ofthe FGF-CX and/or FCTRX ligand/FGF-CX and/or FCTRX interaction may beuseful therapeutically for both the treatment of proliferative anddifferentiative disorders, as well as modulating (e.g. promoting orinhibiting) cell survival. Moreover, the FGF-CX and/orFCTRX-immunoglobulin fusion proteins of the invention can be used asimmunogens to produce anti-FGF-CX and/or FCTRX antibodies in a subject,to purify FGF-CX and/or FCTRX ligands, and in screening assays toidentify molecules that inhibit the interaction of FGF-CX and/or FCTRXwith an FGF-CX and/or FCTRX ligand.

An FGF-CX and/or FCTRX chimeric or fusion protein of the invention canbe produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, e.g., byemploying blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers that give rise tocomplementary overhangs between two consecutive gene fragments that cansubsequently be annealed and reamplified to generate a chimeric genesequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST polypeptide). An FGF-CX and/or FCTRX-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the FGF-CX and/or FCTRX protein.

FGF-CX and/or FCTRX Agonists and Antagonists

The invention also pertains to variants of the FGF-CX and/or FCTRXproteins that function as either FGF-CX and/or FCTRX agonists (i.e.,mimetics) or as FGF-CX and/or FCTRX antagonists. Variants of the FGF-CXand/or FCTRX protein can be generated by mutagenesis (e.g., discretepoint mutation or truncation of the FGF-CX and/or FCTRX protein). Anagonist of the FGF-CX and/or FCTRX protein can retain substantially thesame, or a subset of, the biological activities of the naturallyoccurring form of the FGF-CX and/or FCTRX protein. An antagonist of theFGF-CX and/or FCTRX protein can inhibit one or more of the activities ofthe naturally occurring form of the FGF-CX and/or FCTRX protein by, forexample, competitively binding to a downstream or upstream member of acellular signaling cascade which includes the FGF-CX and/or FCTRXprotein. Thus, specific biological effects can be elicited by treatmentwith a variant of limited function. In one embodiment, treatment of asubject with a variant having a subset of the biological activities ofthe naturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of theFGF-CX and/or FCTRX proteins.

Variants of the FGF-CX and/or FCTRX proteins that function as eitherFGF-CX and/or FCTRX agonists (i.e., mimetics) or as FGF-CX and/or FCTRXantagonists can be identified by screening combinatorial libraries ofmutants (e.g., truncation mutants) of the FGF-CX and/or FCTRX proteinsfor FGF-CX and/or FCTRX protein agonist or antagonist activity. In oneembodiment, a variegated library of FGF-CX and/or FCTRX variants isgenerated by combinatorial mutagenesis at the nucleic acid level and isencoded by a variegated gene library. A variegated library of FGF-CXand/or FCTRX variants can be produced by, for example, enzymaticallyligating a mixture of synthetic oligonucleotides into gene sequencessuch that a degenerate set of potential FGF-CX and/or FCTRX sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofFGF-CX and/or FCTRX sequences therein. There are a variety of methodswhich can be used to produce libraries of potential FGF-CX and/or FCTRXvariants from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be performed in an automatic DNAsynthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential FGF-CX and/or FCTRX sequences. Methods for synthesizingdegenerate oligonucleotides are well-known within the art. See, e.g.,Narang, 1983. Tetrahedron 39: 3; Itakura, et al., 1984. Annu. Rev.Biochem. 53: 323; Itakura, et al., 1984. Science 198: 1056; Ike, et al.,1983. Nucl. Acids Res. 11: 477.

Polypeptide Libraries

In addition, libraries of fragments of the FGF-CX and/or FCTRX proteincoding sequences can be used to generate a variegated population ofFGF-CX and/or FCTRX fragments for screening and subsequent selection ofvariants of an FGF-CX and/or FCTRX protein. In one embodiment, a libraryof coding sequence fragments can be generated by treating a doublestranded PCR fragment of an FGF-CX and/or FCTRX coding sequence with anuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble-stranded DNA that can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S₁ nuclease, and ligating theresulting fragment library into an expression vector. By this method,expression libraries can be derived which encodes N-terminal andinternal fragments of various sizes of the FGF-CX and/or FCTRX proteins.

Various techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of FGF-CX and/or FCTRXproteins. The most widely used techniques, which are amenable to highthroughput analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a new technique that enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify FGF-CX and/or FCTRX variants. See, e.g.,Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815;Delgrave, et al., 1993. Protein Engineering 6:327-331.

Anti-FGF-CX and/or FCTRX Antibodies

Also included in the invention are antibodies to FGF-CX and/or FCTRXproteins, or fragments of FGF-CX and/or FCTRX proteins. The term“antibody” as used herein refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin (Ig) molecules, i.e.,molecules that contain an antigen binding site that specifically binds(immunoreacts with) an antigen. Such antibodies include, but are notlimited to, polyclonal, monoclonal, chimeric, single chain, F_(ab),F_(ab′) and F_((ab′)2) fragments, and an F_(ab) expression library. Ingeneral, an antibody molecule obtained from humans relates to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.Reference herein to antibodies includes a reference to all such classes,subclasses and types of human antibody species.

An isolated FGF-CX and/or FCTRX-related protein of the invention may beintended to serve as an antigen, or a portion or fragment thereof, andadditionally can be used as an immunogen to generate antibodies thatimmunospecifically bind the antigen, using standard techniques forpolyclonal and monoclonal antibody preparation. The full-length proteincan be used or, alternatively, the invention provides antigenic peptidefragments of the antigen for use as immunogens. An antigenic peptidefragment comprises at least 6 amino acid residues of the amino acidsequence of the full length protein and encompasses an epitope thereofsuch that an antibody raised against the peptide forms a specific immunecomplex with the full length protein or with any fragment that containsthe epitope. Preferably, the antigenic peptide comprises at least 10amino acid residues, or at least 15 amino acid residues, or at least 20amino acid residues, or at least 30 amino acid residues. Preferredepitopes encompassed by the antigenic peptide are regions of the proteinthat are located on its surface; commonly these are hydrophilic regions.

In certain embodiments of the invention, at least one epitopeencompassed by the antigenic peptide is a region of FGF-CX and/orFCTRX-related protein that is located on the surface of the protein,e.g., a hydrophilic region. A hydrophobicity analysis of the humanFGF-CX and/or FCTRX-related protein sequence will indicate which regionsof a FGF-CX and/or FCTRX-related protein are particularly hydrophilicand, therefore, are likely to encode surface residues useful fortargeting antibody production. As a means for targeting antibodyproduction, hydropathy plots showing regions of hydrophilicity andhydrophobicity may be generated by any method well known in the art,including, for example, the Kyte Doolittle or the Hopp Woods methods,either with or without Fourier transformation. See, e.g., Hopp andWoods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle1982, J. Mol. Biol. 157: 105-142, each of which is incorporated hereinby reference in its entirety. Antibodies that are specific for one ormore domains within an antigenic protein, or derivatives, fragments,analogs or homologs thereof, are also provided herein.

A protein of the invention, or a derivative, fragment, analog, homologor ortholog thereof, may be utilized as an immunogen in the generationof antibodies that immunospecifically bind these protein components.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against a protein of theinvention, or against derivatives, fragments, analogs homologs ororthologs thereof (see, for example, Antibodies: A Laboratory Manual,Harlow and Lane, 1988, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., incorporated herein by reference). Some of theseantibodies are discussed below.

Polyclonal Antibodies

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byone or more injections with the native protein, a synthetic variantthereof, or a derivative of the foregoing. An appropriate immunogenicpreparation can contain, for example, the naturally occurringimmunogenic protein, a chemically synthesized polypeptide representingthe immunogenic protein, or a recombinantly expressed immunogenicprotein. Furthermore, the protein may be conjugated to a second proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. The preparation can further include an adjuvant. Variousadjuvants used to increase the immunological response include, but arenot limited to, Freund's (complete and incomplete), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol,etc.), adjuvants usable in humans such as Bacille Calmette-Guerin andCorynebacterium parvum, or similar immunostimulatory agents. Additionalexamples of adjuvants which can be employed include MPL-TDM adjuvant(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

The polyclonal antibody molecules directed against the immunogenicprotein can be isolated from the mammal (e.g., from the blood) andfurther purified by well known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

Monoclonal Antibodies

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs thus contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes can beimmunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MONOCLONALANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., MONOCLONAL ANTIBODY PRODUCTION TECHNIQUES AND APPLICATIONS, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Preferably, antibodies having ahigh degree of specificity and a high binding affinity for the targetantigen are isolated.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.Suitable culture media for this purpose include, for example, Dulbecco'sModified Eagle's Medium and RPMI-1640 medium. Alternatively, thehybridoma cells can be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies can also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

Humanized Antibodies

The antibodies directed against the protein antigens of the inventioncan further comprise humanized antibodies or human antibodies. Theseantibodies are suitable for administration to humans without engenderingan immune response by the human against the administered immunoglobulin.Humanized forms of antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) that areprincipally comprised of the sequence of a human immunoglobulin, andcontain minimal sequence derived from a non-human immunoglobulin.Humanization can be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. (See also U.S. Pat.No.5,225,539.) In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies can also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin consensussequence. The humanized antibody optimally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

Human Antibodies

Fully human antibodies relate to antibody molecules in which essentiallythe entire sequences of both the light chain and the heavy chain,including the CDRs, arise from human genes. Such antibodies are termed“human antibodies”, or “fully human antibodies” herein. Human monoclonalantibodies can be prepared by the trioma technique; the human B-cellhybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) andthe EBV hybridoma technique to produce human monoclonal antibodies (seeCole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized inthe practice of the present invention and may be produced by using humanhybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)). Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.(Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859(1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al,(NatureBiotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14,826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93(1995)).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT publication WO94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed inPCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598. It can be obtained by a methodincluding deleting the J segment genes from at least one endogenousheavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearrangedimmunoglobulin heavy chain locus, the deletion being effected by atargeting vector containing a gene encoding a selectable marker; andproducing from the embryonic stem cell a transgenic mouse whose somaticand germ cells contain the gene encoding the selectable marker.

A method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771. It includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying aclinically relevant epitope on an immunogen, and a correlative methodfor selecting an antibody that binds immunospecifically to the relevantepitope with high affinity, are disclosed in PCT publication WO99/53049.

F_(ab) Fragments and Single Chain Antibodies

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to an antigenic protein of theinvention (see e.g., U.S. Pat. No. 4,946,778). In addition, methods canbe adapted for the construction of F_(ab) expression libraries (seee.g., Huse, et al., 1989 Science 246: 1275-128.1) to allow rapid andeffective identification of monoclonal F_(ab) fragments with the desiredspecificity for a protein or derivatives, fragments, analogs or homologsthereof. Antibody fragments that contain the idiotypes to a proteinantigen may be produced by techniques known in the art including, butnot limited to: (i) an F_((ab′)2) fragment produced by pepsin digestionof an antibody molecule; (ii) an F_(ab) fragment generated by reducingthe disulfide bridges of an F_((ab′)2) fragment; (iii) an F_(ab)fragment generated by the treatment of the antibody molecule with papainand a reducing agent and (iv) F_(v) fragments.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is foran antigenic protein of the invention. The second binding target is anyother antigen, and advantageously is a cell-surface protein or receptoror receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., 1991 EMBO J.,10:3655-3659.

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular antigen. Bispecific antibodies can alsobe used to direct cytotoxic agents to cells which express a particularantigen. These antibodies possess an antigen-binding arm and an armwhich binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interestbinds the protein antigen described herein and further binds tissuefactor (TF).

Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP03089). It is contemplated that the antibodies can be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins can beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

Effector Function Engineering

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity can also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and can thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody can be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is in turnconjugated to a cytotoxic agent.

In one embodiment, methods for the screening of antibodies that possessthe desired specificity include, but are not limited to, enzyme-linkedimmunosorbent assay (ELISA) and other immunologically-mediatedtechniques known within the art. In a specific embodiment, selection ofantibodies that are specific to a particular domain of an FGF-CX and/orFCTRX protein is facilitated by generation of hybridomas that bind tothe fragment of an FGF-CX and/or FCTRX protein possessing such a domain.Thus, antibodies that are specific for a desired domain within an FGF-CXand/or FCTRX protein, or derivatives, fragments, analogs or homologsthereof, are also provided herein.

Anti-FGF-CX and/or FCTRX antibodies may be used in methods known withinthe art relating to the localization and/or quantitation of an FGF-CXand/or FCTRX protein (e.g., for use in measuring levels of the FGF-CXand/or FCTRX protein within appropriate physiological samples, for usein diagnostic methods, for use in imaging the protein, and the like). Ina given embodiment, antibodies for FGF-CX and/or FCTRX proteins, orderivatives, fragments, analogs or homologs thereof, that contain theantibody derived binding domain, are utilized aspharmacologically-active compounds (hereinafter “Therapeutics”).

An anti-FGF-CX and/or FCTRX antibody (e.g., monoclonal antibody) can beused to isolate an FGF-CX and/or FCTRX polypeptide by standardtechniques, such as affinity chromatography or immunoprecipitation. Ananti-FGF-CX and/or FCTRX antibody can facilitate the purification ofnatural FGF-CX and/or FCTRX polypeptide from cells and ofrecombinantly-produced FGF-CX and/or FCTRX polypeptide expressed in hostcells. Moreover, an anti-FGF-CX and/or FCTRX antibody can be used todetect FGF-CX and/or FCTRX protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the FGF-CX and/or FCTRX protein. Anti-FGF-CX and/or FCTRXantibodies can be used diagnostically to monitor protein levels intissue as part of a clinical testing procedure, e.g., to, for example,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

FGF-CX and/or FCTRX Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding an FGF-CX and/orFCTRX protein, or derivatives, fragments, analogs or homologs thereof.As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively-linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably-linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell).

The term “regulatory sequence” is intended to includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cell and those that direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or peptides, including fusion proteinsor peptides, encoded by nucleic acids as described herein (e.g., FGF-CXand/or FCTRX proteins, mutant forms of FGF-CX and/or FCTRX proteins,fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of FGF-CX and/or FCTRX proteins in prokaryotic or eukaryoticcells. For example, FGF-CX and/or FCTRX proteins can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out inEscherichia coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionproteins. Fusion vectors add a number of amino acids to a proteinencoded therein, usually to the amino terminus of the recombinantprotein. Such fusion vectors typically serve three purposes: (i) toincrease expression of recombinant protein; (ii) to increase thesolubility of the recombinant protein; and (iii) to aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, e.g., Gottesman,GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,San Diego, Calif. (1990) 119-128. Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (see, e.g., Wada, et al., 1992.Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acidsequences of the invention can be carried out by standard DNA synthesistechniques.

In another embodiment, the FGF-CX and/or FCTRX expression vector is ayeast expression vector. Examples of vectors for expression in yeastSaccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J.6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943),pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,Calif.).

Alternatively, FGF-CX and/or FCTRX can be expressed in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., SF9 cells)include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840)and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, adenovirus 2, cytomegalovirus, andsimian virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 ofSambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively-linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to FGF-CX and/or FCTRX mRNA. Regulatory sequencesoperatively linked to a nucleic acid cloned in the antisense orientationcan be chosen that direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen that directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see, e.g.,Weintraub, et al., “Antisense RNA as a molecular tool for geneticanalysis,” Reviews-Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,FGF-CX and/or FCTRX protein can be expressed in bacterial cells such asE. coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells are known tothose skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MOLECULARCLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding FGF-CX and/or FCTRX or can be introduced on a separate vector.Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) FGF-CX and/orFCTRX protein. Accordingly, the invention further provides methods forproducing FGF-CX and/or FCTRX protein using the host cells of theinvention. In one embodiment, the method comprises culturing the hostcell of invention (into which a recombinant expression vector encodingFGF-CX and/or FCTRX protein has been introduced) in a suitable mediumsuch that FGF-CX and/or FCTRX protein is produced. In anotherembodiment, the method further comprises isolating FGF-CX and/or FCTRXprotein from the medium or the host cell.

Transgenic FGF-CX and/or FCTRX Animals

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichFGF-CX and/or FCTRX protein-coding sequences have been introduced. Suchhost cells can then be used to create non-human transgenic animals inwhich exogenous FGF-CX and/or FCTRX sequences have been introduced intotheir genome or homologous recombinant animals in which endogenousFGF-CX and/or FCTRX sequences have been altered. Such animals are usefulfor studying the function and/or activity of FGF-CX and/or FCTRX proteinand for identifying and/or evaluating modulators of FGF-CX and/or FCTRXprotein activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA that is integrated into the genome of a cellfrom which a transgenic animal develops and that remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, a “homologous recombinant animal” is a non-humananimal, preferably a mammal, more preferably a mouse, in which anendogenous FGF-CX and/or FCTRX gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

A transgenic animal of the invention can be created by introducingFGF-CX and/or FCTRX-encoding nucleic acid into the male pronuclei of afertilized oocyte (e.g., by microinjection, retroviral infection) andallowing the oocyte to develop in a pseudopregnant female foster animal.The human FGF-CX and/or FCTRX cDNA sequences of SEQ ID NOS:1, 3, 5, 7,9, 11 and 13 can be introduced as a transgene into the genome of anon-human animal. Alternatively, a non-human homologue of the humanFGF-CX and/or FCTRX gene, such as a mouse FGF-CX and/or FCTRX gene, canbe isolated based on hybridization to the human FGF-CX and/or FCTRX cDNA(described further supra) and used as a transgene. Intronic sequencesand polyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably-linked to theFGF-CX and/or FCTRX transgene to direct expression of FGF-CX and/orFCTRX protein to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866; 4,870,009; and 4,873,191; andHogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. Similar methods are used forproduction of other transgenic animals. A transgenic founder animal canbe identified based upon the presence of the FGF-CX and/or FCTRXtransgene in its genome and/or expression of FGF-CX and/or FCTRX mRNA intissues or cells of the animals. A transgenic founder animal can then beused to breed additional animals carrying the transgene. Moreover,transgenic animals carrying a transgene-encoding FGF-CX and/or FCTRXprotein can further be bred to other transgenic animals carrying othertransgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of an FGF-CX and/or FCTRX gene into which adeletion, addition or substitution has been introduced to thereby alter,e.g., functionally disrupt, the FGF-CX and/or FCTRX gene. The FGF-CXand/or FCTRX gene can be a human gene (e.g., the cDNA of SEQ ID NOS:1,3, 5, 7, 9, 11 and 13), but more preferably, is a non-human homologue ofa human FGF-CX and/or FCTRX gene. For example, a mouse homologue ofhuman FGF-CX and/or FCTRX gene of SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13can be used to construct a homologous recombination vector suitable foraltering an endogenous FGF-CX and/or FCTRX gene in the mouse genome. Inone embodiment, the vector is designed such that, upon homologousrecombination, the endogenous FGF-CX and/or FCTRX gene is functionallydisrupted (i.e., no longer encodes a functional protein; also referredto as a “knock out” vector).

Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous FGF-CX and/or FCTRX gene is mutated orotherwise altered but still encodes functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous FGF-CX and/or FCTRX protein). In thehomologous recombination vector, the altered portion of the FGF-CXand/or FCTRX gene is flanked at its 5′- and 3′-termini by additionalnucleic acid of the FGF-CX and/or FCTRX gene to allow for homologousrecombination to occur between the exogenous FGF-CX and/or FCTRX genecarried by the vector and an endogenous FGF-CX and/or FCTRX gene in anembryonic stem cell. The additional flanking FGF-CX and/or FCTRX nucleicacid is of sufficient length for successful homologous recombinationwith the endogenous gene. Typically, several kilobases of flanking DNA(both at the 5′- and 3′-termini) are included in the vector. See, e.g.,Thomas, et al., 1987. Cell 51: 503 for a description of homologousrecombination vectors. The vector is ten introduced into an embryonicstem cell line (e.g., by electroporation) and cells in which theintroduced FGF-CX and/or FCTRX gene has homologously-recombined with theendogenous FGF-CX and/or FCTRX gene are selected. See, e.g., Li, et al.,1992. Cell 69: 915.

The selected cells are then injected into a blastocyst of an animal(e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987.In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH,Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously-recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously-recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley, 1991. Curr. Opin. Biotechnol. 2: 823-829; PCTInternational Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968;and WO 93/04169.

In another embodiment, transgenic non-humans animals can be producedthat contain selected systems that allow for regulated expression of thetransgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, See, e.g., Lakso, et al., 1992. Proc. Natl. Acad.Sci. USA 89: 6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, etal., 1991. Science 251:1351-1355. If a cre/loxP recombinase system isused to regulate expression of the transgene, animals containingtransgenes encoding both the Cre recombinase and a selected protein arerequired. Such animals can be provided through the construction of“double” transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, et al., 1997.Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G₀ phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyte and then transferred to pseudopregnant female foster animal.The offspring borne of this female foster animal will be a clone of theanimal from which the cell (e.g., the somatic cell) is isolated.

Pharmaceutical Compositions

The FGF-CX and/or FCTRX nucleic acid molecules, FGF-CX and/or FCTRXproteins, and anti-FGF-CX and/or FCTRX antibodies (also referred toherein as “active compounds”) of the invention, and derivatives,fragments, analogs and homologs thereof, can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, finger's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., an FGF-CX and/or FCTRX protein or anti-FGF-CX and/orFCTRX antibody) in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, methods of preparation arevacuum drying and freeze-drying that yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotacticinjection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Screening and Detection Methods

The isolated nucleic acid molecules of the invention can be used toexpress FGF-CX and/or FCTRX protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect FGF-CXand/or FCTRX mRNA (e.g., in a biological sample) or a genetic lesion inan FGF-CX and/or FCTRX gene, and to modulate FGF-CX and/or FCTRXactivity, as described further, below. In addition, the FGF-CX and/orFCTRX proteins can be used to screen drugs or compounds that modulatethe FGF-CX and/or FCTRX protein activity or expression as well as totreat disorders characterized by insufficient or excessive production ofFGF-CX and/or FCTRX protein or production of FGF-CX and/or FCTRX proteinforms that have decreased or aberrant activity compared to FGF-CX and/orFCTRX wild-type protein (e.g.; diabetes (regulates insulin release);obesity (binds and transport lipids); metabolic disturbances associatedwith obesity, the metabolic syndrome X as well as anorexia and wastingdisorders associated with chronic diseases and various cancers, andinfectious disease(possesses anti-microbial activity) and the variousdyslipidemias. In addition, the anti-FGF-CX and/or FCTRX antibodies ofthe invention can be used to detect and isolate FGF-CX and/or FCTRXproteins and modulate FGF-CX and/or FCTRX activity. In yet a furtheraspect, the invention can be used in methods to influence appetite,absorption of nutrients and the disposition of metabolic substrates inboth a positive and negative fashion.

The invention further pertains to novel agents identified by thescreening assays described herein and uses thereof for treatments asdescribed, supra.

Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)that bind to FGF-CX and/or FCTRX proteins or have a stimulatory orinhibitory effect on, e.g., FGF-CX and/or FCTRX protein expression orFGF-CX and/or FCTRX protein activity. The invention also includescompounds identified in the screening assays described herein.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of themembrane-bound form of an FGF-CX and/or FCTRX protein or polypeptide orbiologically-active portion thereof. The test compounds of the inventioncan be obtained using any of the numerous approaches in combinatoriallibrary methods known in the art, including: biological libraries;spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the “one-beadone-compound” library method; and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds. See, e.g., Lam, 1997. Anticancer Drug Design 12: 145.

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules. Libraries of chemical and/or biologicalmixtures, such as fungal, bacterial, or algal extracts, are known in theart and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al., 1993. Proc. Natl.Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci.U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho,et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed.Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354:82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner, U.S. Pat. No. 5,233,409),plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89: 1865-1869)or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990.Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci.U.S.A. 87: 6378-6382; Felici, 1991. J. Mol Biol. 222: 301-310; Ladner,U.S. Pat. No. 5,233,409.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a membrane-bound form of FGF-CX and/or FCTRX protein, or abiologically-active portion thereof, on the cell surface is contactedwith a test compound and the ability of the test compound to bind to anFGF-CX and/or FCTRX protein determined. The cell, for example, can ofmammalian origin or a yeast cell. Determining the ability of the testcompound to bind to the FGF-CX and/or FCTRX protein can be accomplished,for example, by coupling the test compound with a radioisotope orenzymatic label such that binding of the test compound to the FGF-CXand/or FCTRX protein or biologically-active portion thereof can bedetermined by detecting the labeled compound in a complex. For example,test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, eitherdirectly or indirectly, and the radioisotope detected by direct countingof radioemission or by scintillation counting. Alternatively, testcompounds can be enzymatically-labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct. In one embodiment, the assay comprises contacting a cell whichexpresses a membrane-bound form of FGF-CX and/or FCTRX protein, or abiologically-active portion thereof, on the cell surface with a knowncompound which binds FGF-CX and/or FCTRX to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with an FGF-CX and/or FCTRXprotein, wherein determining the ability of the test compound tointeract with an FGF-CX and/or FCTRX protein comprises determining theability of the test compound to preferentially bind to FGF-CX and/orFCTRX protein or a biologically-active portion thereof as compared tothe known compound.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of FGF-CX and/orFCTRX protein, or a biologically-active portion thereof, on the cellsurface with a test compound and determining the ability of the testcompound to modulate (e.g., stimulate or inhibit) the activity of theFGF-CX and/or FCTRX protein or biologically-active portion thereof.Determining the ability of the test compound to modulate the activity ofFGF-CX and/or FCTRX or a biologically-active portion thereof can beaccomplished, for example, by determining the ability of the FGF-CXand/or FCTRX protein to bind to or interact with an FGF-CX and/or FCTRXtarget molecule. As used herein, a “target molecule” is a molecule withwhich an FGF-CX and/or FCTRX protein binds or interacts in nature, forexample, a molecule on the surface of a cell which expresses an FGF-CXand/or FCTRX interacting protein, a molecule on the surface of a secondcell, a molecule in the extracellular milieu, a molecule associated withthe internal surface of a cell membrane or a cytoplasmic molecule. AnFGF-CX and/or FCTRX target molecule can be a non-FGF-CX and/or FCTRXmolecule or an FGF-CX and/or FCTRX protein or polypeptide of theinvention. In one embodiment, an FGF-CX and/or FCTRX target molecule isa component of a signal transduction pathway that facilitatestransduction of an extracellular signal (e.g. a signal generated bybinding of a compound to a membrane-bound FGF-CX and/or FCTRX molecule)through the cell membrane and into the cell. The target, for example,can be a second intercellular protein that has catalytic activity or aprotein that facilitates the association of downstream signalingmolecules with FGF-CX and/or FCTRX.

Determining the ability of the FGF-CX and/or FCTRX protein to bind to orinteract with an FGF-CX and/or FCTRX target molecule can be accomplishedby one of the methods described above for determining direct binding. Inone embodiment, determining the ability of the FGF-CX and/or FCTRXprotein to bind to or interact with an FGF-CX and/or FCTRX targetmolecule can be accomplished by determining the activity of the targetmolecule. For example, the activity of the target molecule can bedetermined by detecting induction of a cellular second messenger of thetarget (i.e. intracellular Ca²⁺, diacylglycerol, IP₃, etc.), detectingcatalytic/enzymatic activity of the target an appropriate substrate,detecting the induction of a reporter gene (comprising an FGF-CX and/orFCTRX-responsive regulatory element operatively linked to a nucleic acidencoding a detectable marker, e.g., luciferase), or detecting a cellularresponse, for example, cell survival, cellular differentiation, or cellproliferation.

In yet another embodiment, an assay of the invention is a cell-freeassay comprising contacting an FGF-CX and/or FCTRX protein orbiologically-active portion thereof with a test compound and determiningthe ability of the test compound to bind to the FGF-CX and/or FCTRXprotein or biologically-active portion thereof. Binding of the testcompound to the FGF-CX and/or FCTRX protein can be determined eitherdirectly or indirectly as described above. In one such embodiment, theassay comprises contacting the FGF-CX and/or FCTRX protein orbiologically-active portion thereof with a known compound which bindsFGF-CX and/or FCTRX to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with an FGF-CX and/or FCTRX protein, wherein determining the ability of the test compound to interact with an FGF-CXand/or FCTRX protein comprises determining the ability of the testcompound to preferentially bind to FGF-CX and/or FCTRX orbiologically-active portion thereof as compared to the known compound.

In still another embodiment, an assay is a cell-free assay comprisingcontacting FGF-CX and/or FCTRX protein or biologically-active portionthereof with a test compound and determining the ability of the testcompound to modulate (e.g. stimulate or inhibit) the activity of theFGF-CX and/or FCTRX protein or biologically-active portion thereof.Determining the ability of the test compound to modulate the activity ofFGF-CX and/or FCTRX can be accomplished, for example, by determining theability of the FGF-CX and/or FCTRX protein to bind to an FGF-CX and/orFCTRX target molecule by one of the methods described above fordetermining direct binding. In an alternative embodiment, determiningthe ability of the test compound to modulate the activity of FGF-CXand/or FCTRX protein can be accomplished by determining the ability ofthe FGF-CX and/or FCTRX protein further modulate an FGF-CX and/or FCTRXtarget molecule. For example, the catalytic/enzymatic activity of thetarget molecule on an appropriate substrate can be determined asdescribed, supra.

In yet another embodiment, the cell-free assay comprises contacting theFGF-CX and/or FCTRX protein or biologically-active portion thereof witha known compound which binds FGF-CX and/or FCTRX protein to form anassay mixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with an FGF-CXand/or FCTRX protein, wherein determining the ability of the testcompound to interact with an FGF-CX and/or FCTRX protein comprisesdetermining the ability of the FGF-CX and/or FCTRX protein topreferentially bind to or modulate the activity of an FGF-CX and/orFCTRX target molecule.

The cell-free assays of the invention are amenable to use of both thesoluble form or the membrane-bound form of FGF-CX and/or FCTRX protein.In the case of cell-free assays comprising the membrane-bound form ofFGF-CX and/or FCTRX protein, it may be desirable to utilize asolubilizing agent such that the membrane-bound form of FGF-CX and/orFCTRX protein is maintained in solution. Examples of such solubilizingagents include non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO).

In more than one embodiment of the above assay methods of the invention,it may be desirable to immobilize either FGF-CX and/or FCTRX protein orits target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound toFGF-CX and/or FCTRX protein, or interaction of FGF-CX and/or FCTRXprotein with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided that adds a domain that allows one orboth of the proteins to be bound to a matrix. For example, GST-FGF-CXand/or FCTRX fusion proteins or GST-target fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtiter plates, that are thencombined with the test compound or the test compound and either thenon-adsorbed target protein or FGF-CX and/or FCTRX protein, and themixture is incubated under conditions conducive to complex formation(e.g., at physiological conditions for salt and pH). Followingincubation, the beads or microtiter plate wells are washed to remove anyunbound components, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as described,supra. Alternatively, the complexes can be dissociated from the matrix,and the level of FGF-CX and/or FCTRX protein binding or activitydetermined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either the FGF-CXand/or FCTRX protein or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated FGF-CX and/or FCTRXprotein or target molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well-known within the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with FGF-CX and/or FCTRXprotein or target molecules, but which do not interfere with binding ofthe FGF-CX and/or FCTRX protein to its target molecule, can bederivatized to the wells of the plate, and unbound target or FGF-CXand/or FCTRX protein trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the FGF-CX and/or FCTRX proteinor target molecule, as well as enzyme-linked assays that rely ondetecting an enzymatic activity associated with the FGF-CX and/or FCTRXprotein or target molecule.

In another embodiment, modulators of FGF-CX and/or FCTRX proteinexpression are identified in a method wherein a cell is contacted with acandidate compound and the expression of FGF-CX and/or FCTRX mRNA orprotein in the cell is determined. The level of expression of FGF-CXand/or FCTRX mRNA or protein in the presence of the candidate compoundis compared to the level of expression of FGF-CX and/or FCTRX mRNA orprotein in the absence of the candidate compound. The candidate compoundcan then be identified as a modulator of FGF-CX and/or FCTRX mRNA orprotein expression based upon this comparison. For example, whenexpression of FGF-CX and/or FCTRX mRNA or protein is greater (i.e.,statistically significantly greater) in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of FGF-CX and/or FCTRX mRNA or protein expression.Alternatively, when expression of FGF-CX and/or FCTRX mRNA or protein isless (statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of FGF-CX and/or FCTRX mRNA or protein expression. The levelof FGF-CX and/or FCTRX mRNA or protein expression in the cells can bedetermined by methods described herein for detecting FGF-CX and/or FCTRXmRNA or protein.

In yet another aspect of the invention, the FGF-CX and/or FCTRX proteinscan be used as “bait proteins” in a two-hybrid assay or three hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos, et al., 1993. Cell72: 223-232; Madura, et al., 1993. J. Biol. Chem. 268: 12046-12054;Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993.Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify otherproteins that bind to or interact with FGF-CX and/or FCTRX (“FGF-CXand/or FCTRX-binding proteins” or “FGF-CX and/or FCTRX-bp”) and modulateFGF-CX and/or FCTRX activity. Such FGF-CX and/or FCTRX-binding proteinsare also likely to be involved in the propagation of signals by theFGF-CX and/or FCTRX proteins as, for example, upstream or downstreamelements of the FGF-CX and/or FCTRX pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for FGF-CX and/orFCTRX is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming an FGF-CXand/or FCTRX-dependent complex, the DNA-binding and activation domainsof the transcription factor are brought into close proximity. Thisproximity allows transcription of a reporter gene (e.g., LacZ) that isoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene that encodes the proteinwhich interacts with FGF-CX and/or FCTRX.

The invention further pertains to novel agents identified by theaforementioned screening assays and uses thereof for treatments asdescribed herein.

Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. By way of example, and not of limitation, thesesequences can be used to: (i) map their respective genes on achromosome; and, thus, locate gene regions associated with geneticdisease; (ii) identify an individual from a minute biological sample(tissue typing); and (iii) aid in forensic identification of abiological sample. Some of these applications are described in thesubsections, below.

Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the FGF-CX and/or FCTRX sequences, SEQ IDNOS:1, 3, 5, 7, 9, 11 and 13, or fragments or derivatives thereof, canbe used to map the location of the FGF-CX and/or FCTRX genes,respectively, on a chromosome. The mapping of the FGF-CX and/or FCTRXsequences to chromosomes is an important first step in correlating thesesequences with genes associated with disease.

Briefly, FGF-CX and/or FCTRX genes can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp in length) from the FGF-CXand/or FCTRX sequences. Computer analysis of the FGF-CX and/or FCTRX,sequences can be used to rapidly select primers that do not span morethan one exon in the genomic DNA, thus complicating the amplificationprocess. These primers can then be used for PCR screening of somaticcell hybrids containing individual human chromosomes. Only those hybridscontaining the human gene corresponding to the FGF-CX and/or FCTRXsequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but in whichhuman cells can, the one human chromosome that contains the geneencoding the needed enzyme will be retained. By using various media,panels of hybrid cell lines can be established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes, and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes. See, e.g.,D'Eustachio, et al., 1983. Science 220: 919-924. Somatic cell hybridscontaining only fragments of human chromosomes can also be produced byusing human chromosomes with translocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the FGF-CXand/or FCTRX sequences to design oligonucleotide primers,sub-localization can be achieved with panels of fragments from specificchromosomes.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical likecolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases, willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see, Verma, et al., HUMAN CHROMOSOMES: A MANUAL OFBASIC TECHNIQUES (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, e.g., in McKusick, MENDELIANINHERITANCE IN MAN, available on-line through Johns Hopkins UniversityWelch Medical Library). The relationship between genes and disease,mapped to the same chromosomal region, can then be identified throughlinkage analysis (co-inheritance of physically adjacent genes),described in, e.g., Egeland, et al., 1987. Nature, 325: 783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the FGF-CX and/or FCTRXgene, can be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

Tissue Typing

The FGF-CX and/or FCTRX sequences of the invention can also be used toidentify individuals from minute biological samples. In this technique,an individual's genomic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentification. The sequences of the invention are useful as additionalDNA markers for RFLP (“restriction fragment length polymorphisms,”described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the invention can be used to provide analternative technique that determines the actual base-by-base DNAsequence of selected portions of an individual's genome. Thus, theFGF-CX and/or FCTRX sequences described herein can be used to preparetwo PCR primers from the 5′- and 3′-termini of the sequences. Theseprimers can then be used to amplify an individual's DNA and subsequentlysequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the invention can be used to obtain suchidentification sequences from individuals and from tissue. The FGF-CXand/or FCTRX sequences of the invention uniquely represent portions ofthe human genome. Allelic variation occurs to some degree in the codingregions of these sequences, and to a greater degree in the noncodingregions. It is estimated that allelic variation between individualhumans occurs with a frequency of about once per each 500 bases. Much ofthe allelic variation is due to single nucleotide polymorphisms (SNPs),which include restriction fragment length polymorphisms (RFLPs).

Each of the sequences described herein can, to some degree, be used as astandard against which DNA from an individual can be compared foridentification purposes. Because greater numbers of polymorphisms occurin the noncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences can comfortably provide positiveindividual identification with a panel of perhaps 10 to 1,000 primersthat each yield a noncoding amplified sequence of 100 bases. Ifpredicted coding sequences, such as those in SEQ ID NOS:1, 3, 5, 7, 9,11 and 13 are used, a more appropriate number of primers for positiveindividual identification would be 500-2,000.

Predictive Medicine

The invention also pertains to the field of predictive medicine in whichdiagnostic assays, prognostic assays, pharmacogenomics, and monitoringclinical trials are used for prognostic (predictive) purposes to therebytreat an individual prophylactically. Accordingly, one aspect of theinvention relates to diagnostic assays for determining FGF-CX and/orFCTRX protein and/or nucleic acid expression as well as FGF-CX and/orFCTRX activity, in the context of a biological sample (e.g., blood,serum, cells, tissue) to thereby determine whether an individual isafflicted with a disease or disorder, or is at risk of developing adisorder, associated with aberrant FGF-CX expression or activity,aberrant FCTRX expression or activity, or both. The disorders includepathology such as inflammatory conditions in the gastrointestinal tract,including but not limited to inflammatory bowel disease such asulcerative colitis and Crohn's disease, growth and proliferativediseases such as cancer, angiogenesis, atherosclerotic plaques, collagenformation, cartilage and bone formation, cardiovascular and fibroticdiseases and diabetic ulcers. In addition, FCTRX nucleic acids and theirencoded polypeptides will be therapeutically useful for the preventionof aneurysms and the acceleration of wound closure through gene therapy.Furthermore, FCTRX nucleic acids and their encoded polypeptides can beutilized to stimulate cellular growth, wound healing, neovascularizationand tissue growth, and similar tissue regeneration needs. Morespecifically, a FCTRX nucleic acid or polypeptide may be useful intreatment of anemia and leukopenia, intestinal tract sensitivity andbaldness. Treatment of such conditions may be indicated, e.g., inpatients having undergone radiation or chemotherapy, wherein treatmentwould minimize any hyperproliferative side effects.

The invention also provides for prognostic (or predictive) assays fordetermining whether an individual is at risk of developing a disorderassociated with FGF-CX and/or FCTRX protein, nucleic acid expression oractivity. For example, mutations in an FGF-CX and/or FCTRX gene can beassayed in a biological sample. Such assays can be used for prognosticor predictive purpose to thereby prophylactically treat an individualprior to the onset of a disorder characterized by or associated withFGF-CX and/or FCTRX protein, nucleic acid expression, or biologicalactivity.

Another aspect of the invention provides methods for determining FGF-CXand/or FCTRX protein, nucleic acid expression or activity in anindividual to thereby select appropriate therapeutic or prophylacticagents for that individual (referred to herein as “pharmacogenomics”).Pharmacogenomics allows for the selection of agents (e.g., drugs) fortherapeutic or prophylactic treatment of an individual based on thegenotype of the individual (e.g., the genotype of the individualexamined to determine the ability of the individual to respond to aparticular agent.)

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs, compounds) on the expression or activity ofFGF-CX and/or FCTRX in clinical trials.

These and other agents are described in further detail in the followingsections.

Diagnostic Assays

An exemplary method for detecting the presence or absence of FGF-CXand/or FCTRX in a biological sample involves obtaining a biologicalsample from a test subject and contacting the biological sample with acompound or an agent capable of detecting FGF-CX and/or FCTRX protein ornucleic acid (e.g., mRNA, genomic DNA) that encodes FGF-CX and/or FCTRXprotein such that the presence of FGF-CX and/or FCTRX is detected in thebiological sample. An agent for detecting FGF-CX and/or FCTRX mRNA orgenomic DNA is a labeled nucleic acid probe capable of hybridizing toFGF-CX and/or FCTRX mRNA or genomic DNA. The nucleic acid probe can be,for example, a full-length FGF-CX and/or FCTRX nucleic acid, such as thenucleic acid of SEQ ID NOS:1, 3, 5, 7, 9, 11 and 13, or a portionthereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or500 nucleotides in length and sufficient to specifically hybridize understringent conditions to FGF-CX and/or FCTRX mRNA or genomic DNA. Othersuitable probes for use in the diagnostic assays of the invention aredescribed herein.

An agent for detecting FGF-CX and/or FCTRX protein is an antibodycapable of binding to FGF-CX and/or FCTRX protein, preferably anantibody with a detectable label. Antibodies can be polyclonal, or morepreferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,Fab or F(ab′)₂) can be used. The term “labeled”, with regard to theprobe or antibody, is intended to encompass direct labeling of the probeor antibody by coupling (i.e., physically linking) a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with another reagent that is directlylabeled. Examples of indirect labeling include detection of a primaryantibody using a fluorescently-labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.That is, the detection method of the invention can be used to detectFGF-CX and/or FCTRX mRNA, protein, or genomic DNA in a biological samplein vitro as well as in vivo. For example, in vitro techniques fordetection of FGF-CX and/or FCTRX mRNA include Northern hybridizationsand in situ hybridizations. In vitro techniques for detection of FGF-CXand/or FCTRX protein include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations, and immunofluorescence.In vitro techniques for detection of FGF-CX and/or FCTRX genomic DNAinclude Southern hybridizations. Furthermore, in vivo techniques fordetection of FGF-CX and/or FCTRX protein include introducing into asubject a labeled anti-FGF-CX and/or FCTRX antibody. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a peripheral blood leukocytesample isolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting FGF-CX and/or FCTRXprotein, mRNA, or genomic DNA, such that the presence of FGF-CX and/orFCTRX protein, mRNA or genomic DNA is detected in the biological sample,and comparing the presence of FGF-CX and/or FCTRX protein, mRNA orgenomic DNA in the control sample with the presence of FGF-CX and/orFCTRX protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of FGF-CXand/or FCTRX in a biological sample. For example, the kit can comprise:a labeled compound or agent capable of detecting FGF-CX and/or FCTRXprotein or mRNA in a biological sample; means for determining the amountof FGF-CX and/or FCTRX in the sample; and means for comparing the amountof FGF-CX and/or FCTRX in the sample with a standard. The compound oragent can be packaged in a suitable container. The kit can furthercomprise instructions for using the kit to detect FGF-CX and/or FCTRXprotein or nucleic acid.

Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant FGF-CX and/or FCTRX expression or activity. Forexample, the assays described herein, such as the preceding diagnosticassays or the following assays, can be utilized to identify a subjecthaving or at risk of developing a disorder associated with FGF-CX and/orFCTRX protein, nucleic acid expression or activity. Alternatively, theprognostic assays can be utilized to identify a subject having or atrisk for developing a disease or disorder. Thus, the invention providesa method for identifying a disease or disorder associated with aberrantFGF-CX and/or FCTRX expression or activity in which a test sample isobtained from a subject and FGF-CX and/or FCTRX protein or nucleic acid(e.g., mRNA, genomic DNA) is detected, wherein the presence of FGF-CXand/or FCTRX protein or nucleic acid is diagnostic for a subject havingor at risk of developing a disease or disorder associated with aberrantFGF-CX and/or FCTRX expression or activity. As used herein, a “testsample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant FGF-CX and/or FCTRX expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a disorder. Thus, the inventionprovides methods for determining whether a subject can be effectivelytreated with an agent for a disorder associated with aberrant FGF-CXand/or FCTRX expression or activity in which a test sample is obtainedand FGF-CX and/or FCTRX protein or nucleic acid is detected (e.g.,wherein the presence of FGF-CX and/or FCTRX protein or nucleic acid isdiagnostic for a subject that can be administered the agent to treat adisorder associated with aberrant FGF-CX and/or FCTRX expression oractivity).

The methods of the invention can also be used to detect genetic lesionsin an FGF-CX and/or FCTRX gene, thereby determining if a subject withthe lesioned gene is at risk for a disorder characterized by aberrantcell proliferation and/or differentiation. In various embodiments, themethods include detecting, in a sample of cells from the subject, thepresence or absence of a genetic lesion characterized by at least one ofan alteration affecting the integrity of a gene encoding an FGF-CXand/or FCTRX-protein, or the misexpression of the FGF-CX and/or FCTRXgene. For example, such genetic lesions can be detected by ascertainingthe existence of at least one of: (i) a deletion of one or morenucleotides from an FGF-CX and/or FCTRX gene; (ii) an addition of one ormore nucleotides to an FGF-CX and/or FCTRX gene; (iii) a substitution ofone or more nucleotides of an FGF-CX and/or FCTRX gene, (iv) achromosomal rearrangement of an FGF-CX and/or FCTRX gene; (v) analteration in the level of a messenger RNA transcript of an FGF-CXand/or FCTRX gene, (vi) aberrant modification of an FGF-CX and/or FCTRXgene, such as of the methylation pattern of the genomic DNA, (vii) thepresence of a non-wild-type splicing pattern of a messenger RNAtranscript of an FGF-CX and/or FCTRX gene, (viii) a non-wild-type levelof an FGF-CX and/or FCTRX protein, (ix) allelic loss of an FGF-CX and/orFCTRX gene, and (x) inappropriate post-translational modification of anFGF-CX and/or FCTRX protein. As described herein, there are a largenumber of assay techniques known in the art which can be used fordetecting lesions in an FGF-CX and/or FCTRX gene. A preferred biologicalsample is a peripheral blood leukocyte sample isolated by conventionalmeans from a subject. However, any biological sample containingnucleated cells may be used, including, for example, buccal mucosalcells.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran,et al., 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994. Proc.Natl. Acad. Sci. USA 91: 360-364), the latter of which can beparticularly useful for detecting point mutations in the FGF-CX and/orFCTRX-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23: 675-682).This method can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers that specifically hybridize to an FGF-CX and/or FCTRX gene underconditions such that hybridization and amplification of the FGF-CXand/or FCTRX gene (if present) occurs, and detecting the presence orabsence of an amplification product, or detecting the size of theamplification product and comparing the length to a control sample. Itis anticipated that PCR and/or LCR may be desirable to use as apreliminary amplification step in conjunction with any of the techniquesused for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (see, Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (see, Kwoh, et al.,1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Qβ Replicase (see,Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in an FGF-CX and/or FCTRX genefrom a sample cell can be identified by alterations in restrictionenzyme cleavage patterns. For example, sample and control DNA isisolated, amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Pat.No. 5,493,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in FGF-CX and/or FCTRX can beidentified by hybridizing a sample and control nucleic acids, e.g., DNAor RNA, to high-density arrays containing hundreds or thousands ofoligonucleotides probes. See, e.g., Cronin, et al., 1996. Human Mutation7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example,genetic mutations in FGF-CX and/or FCTRX can be identified in twodimensional arrays containing light-generated DNA probes as described inCronin, et al., supra. Briefly, a first hybridization array of probescan be used to scan through long stretches of DNA in a sample andcontrol to identify base changes between the sequences by making lineararrays of sequential overlapping probes. This step allows theidentification of point mutations. This is followed by a secondhybridization array that allows the characterization of specificmutations by using smaller, specialized probe arrays complementary toall variants or mutations detected. Each mutation array is composed ofparallel probe sets, one complementary to the wild-type gene and theother complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the FGF-CX and/orFCTRX gene and detect mutations by comparing the sequence of the sampleFGF-CX and/or FCTRX with the corresponding wild-type (control) sequence.Examples of sequencing reactions include those based on techniquesdeveloped by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is alsocontemplated that any of a variety of automated sequencing procedurescan be utilized when performing the diagnostic assays (see, e.g., Naeve,et al., 1995. Biotechniques 19: 448), including sequencing by massspectrometry (see, e.g., PCT International Publication No. WO 94/16101;Cohen, et al., 1996. Adv. Chromatography 36: 127-162; and Griffin, etal., 1993. Appl. Biochem. Biotechnol. 38: 147-159).

Other methods for detecting mutations in the FGF-CX and/or FCTRX geneinclude methods in which protection from cleavage agents is used todetect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See, e.g.,Myers, et al., 1985. Science 230: 1242. In general, the art technique of“mismatch cleavage” starts by providing heteroduplexes of formed byhybridizing (labeled) RNA or DNA containing the wild-type FGF-CX and/orFCTRX sequence with potentially mutant RNA or DNA obtained from a tissuesample. The double-stranded duplexes are treated with an agent thatcleaves single-stranded regions of the duplex such as which will existdue to basepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S₁ nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, e.g.,Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, etal., 1992. Methods Enzymol. 217: 286-295. In an embodiment, the controlDNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in FGF-CX and/or FCTRX cDNAsobtained from samples of cells. For example, the mutY enzyme of E. colicleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLacells cleaves T at G/T mismatches. See, e.g., Hsu, et al., 1994.Carcinogenesis 15: 1657-1662. According to an exemplary embodiment, aprobe based on an FGF-CX and/or FCTRX sequence, e.g., a wild-type FGF-CXand/or FCTRX sequence, is hybridized to a cDNA or other DNA product froma test cell(s). The duplex is treated with a DNA mismatch repair enzyme,and the cleavage products, if any, can be detected from electrophoresisprotocols or the like. See, e.g., U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in FGF-CX and/or FCTRX genes. For example,single strand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids. See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci.USA: 86: 2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992.Genet. Anal. Tech. Appl. 9: 73-79. Single-stranded DNA fragments ofsample and control FGF-CX and/or FCTRX nucleic acids will be denaturedand allowed to renature. The secondary structure of single-strandednucleic acids varies according to sequence, the resulting alteration inelectrophoretic mobility enables the detection of even a single basechange. The DNA fragments may be labeled or detected with labeledprobes. The sensitivity of the assay may be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In one embodiment, the subject method utilizesheteroduplex analysis to separate double stranded heteroduplex moleculeson the basis of changes in electrophoretic mobility. See, e.g., Keen, etal., 1991. Trends Genet. 7: 5.

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers,et al., 1985. Nature 313: 495. When DGGE is used as the method ofanalysis, DNA will be modified to insure that it does not completelydenature, for example by adding a GC clamp of approximately 40 bp ofhigh-melting GC-rich DNA by PCR. In a further embodiment, a temperaturegradient is used in place of a denaturing gradient to identifydifferences in the mobility of control and sample DNA. See, e.g.,Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditions thatpermit hybridization only if a perfect match is found. See, e.g., Saiki,et al., 1986. Nature 324: 163; Saiki, et al., 1989. Proc. Natl. Acad.Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridizedto PCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology that depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization; see, e.g.,Gibbs, et al., 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme3′-terminus of one primer where, under appropriate conditions, mismatchcan prevent, or reduce polymerase extension (see, e.g., Prossner, 1993.Tibtech. 11: 238). In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection. See, e.g., Gasparini, et al, 1992. Mol. Cell Probes 6: 1. Itis anticipated that in certain embodiments amplification may also beperformed using Taq ligase for amplification. See, e.g., Barany, 1991.Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occuronly if there is a perfect match at the 3′-terminus of the 5′ sequence,making it possible to detect the presence of a known mutation at aspecific site by looking for the presence or absence of amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving an FGF-CX and/or FCTRXgene.

Furthermore, any cell type or tissue, preferably peripheral bloodleukocytes, in which FGF-CX and/or FCTRX is expressed may be utilized inthe prognostic assays described herein. However, any biological samplecontaining nucleated cells may be used, including, for example, buccalmucosal cells.

Pharmacogenomics

Agents, or modulators that have a stimulatory or inhibitory effect onFGF-CX and/or FCTRX activity (e.g., FGF-CX and/or FCTRX geneexpression), as identified by a screening assay described herein can beadministered to individuals to treat (prophylactically ortherapeutically) disorders. The disorders include pathology such asinflammatory conditions in the gastrointestinal tract, including but notlimited to inflammatory bowel disease such as ulcerative colitis andCrohn's disease, growth and proliferative diseases such as cancer,angiogenesis, atherosclerotic plaques, collagen formation, cartilage andbone formation, cardiovascular and fibrotic diseases and diabeticulcers. In addition, FCTRX nucleic acids and their encoded polypeptideswill be therapeutically useful for the prevention of aneurysms and theacceleration of wound closure through gene therapy. Furthermore, FCTRXnucleic acids and their encoded polypeptides can be utilized tostimulate cellular growth. wound healing, neovascularization and tissuegrowth, and similar tissue regeneration needs. More specifically, aFCTRX nucleic acid or polypeptide may be useful in treatment of anemiaand leukopenia, intestinal tract sensitivity and baldness. Treatment ofsuch conditions may be indicated, e.g., in patients having undergoneradiation or chemotherapy, wherein treatment would minimize anyhyperproliferative side effects.

In conjunction with such treatment, the pharmacogenomics (i.e., thestudy of the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) of the individualmay be considered. Differences in metabolism of therapeutics can lead tosevere toxicity or therapeutic failure by altering the relation betweendose and blood concentration of the pharmacologically active drug. Thus,the pharmacogenomics of the individual permits the selection ofeffective agents (e.g., drugs) for prophylactic or therapeutictreatments based on a consideration of the individual's genotype. Suchpharmacogenomics can further be used to determine appropriate dosagesand therapeutic regimens. Accordingly, the activity of FGF-CX and/orFCTRX protein, expression of FGF-CX and/or FCTRX nucleic acid, ormutation content of FGF-CX and/or FCTRX genes in an individual can bedetermined to thereby select appropriate agent(s) for therapeutic orprophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp.Pharmacol. Physiol., 23: 983-985; Linder, 1997. Clin. Chem., 43:254-266. In general, two types of pharmacogenetic conditions can bedifferentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase (G6PD) deficiency is a commoninherited enzymopathy in which the main clinical complication ishemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. At the other extreme are the so called ultra-rapidmetabolizers who do not respond to standard doses. Recently, themolecular basis of ultra-rapid metabolism has been identified to be dueto CYP2D6 gene amplification.

Thus, the activity of FGF-CX and/or FCTRX protein, expression of FGF-CXand/or FCTRX nucleic acid, or mutation content of FGF-CX and/or FCTRXgenes in an individual can be determined to thereby select appropriateagent(s) for therapeutic or prophylactic treatment of the individual. Inaddition, pharmacogenetic studies can be used to apply genotyping ofpolymorphic alleles encoding drug-metabolizing enzymes to theidentification of an individual's drug responsiveness phenotype. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with an FGF-CX and/orFCTRX modulator, such as a modulator identified by one of the exemplaryscreening assays described herein.

Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of FGF-CX and/or FCTRX (e.g., the ability tomodulate aberrant cell proliferation and/or differentiation) can beapplied not only in basic drug screening, but also in clinical trials.For example, the effectiveness of an agent determined by a screeningassay as described herein to increase FGF-CX and/or FCTRX geneexpression, protein levels, or upregulate FGF-CX and/or FCTRX activity,can be monitored in clinical trails of subjects exhibiting decreasedFGF-CX and/or FCTRX gene expression, protein levels, or downregulatedFGF-CX and/or FCTRX activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease FGF-CX and/or FCTRXgene expression, protein levels, or downregulate FGF-CX and/or FCTRXactivity, can be monitored in clinical trails of subjects exhibitingincreased FGF-CX and/or FCTRX gene expression, protein levels, orupregulated FGF-CX and/or FCTRX activity. In such clinical trials, theexpression or activity of FGF-CX and/or FCTRX and, preferably, othergenes that have been implicated in, for example, a cellularproliferation or immune disorder can be used as a “read out” or markersof the immune responsiveness of a particular cell.

By way of example, and not of limitation, genes, including FGF-CX and/orFCTRX, that are modulated in cells by treatment with an agent (e.g.,compound, drug or small molecule) that modulates FGF-CX and/or FCTRXactivity (e.g., identified in a screening assay as described herein) canbe identified. Thus, to study the effect of agents on cellularproliferation disorders, for example, in a clinical trial, cells can beisolated and RNA prepared and analyzed for the levels of expression ofFGF-CX and/or FCTRX and other genes implicated in the disorder. Thelevels of gene expression (i.e., a gene expression pattern) can bequantified by Northern blot analysis or RT-PCR, as described herein, oralternatively by measuring the amount of protein produced, by one of themethods as described herein, or by measuring the levels of activity ofFGF-CX and/or FCTRX or other genes. In this manner, the gene expressionpattern can serve as a marker, indicative of the physiological responseof the cells to the agent. Accordingly, this response state may bedetermined before, and at various points during, treatment of theindividual with the agent.

In one embodiment, the invention provides a method for monitoring theeffectiveness of treatment of a subject with an agent (e.g., an agonist,antagonist, protein, peptide, peptidomimetic, nucleic acid, smallmolecule, or other drug candidate identified by the screening assaysdescribed herein) comprising the steps of (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of an FGF-CX and/or FCTRXprotein, mRNA, or genomic DNA in the preadministration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression or activity of the FGF-CX and/or FCTRXprotein, mRNA, or genomic DNA in the post-administration samples; (v)comparing the level of expression or activity of the FGF-CX and/or FCTRXprotein, mRNA, or genomic DNA in the pre-administration sample with theFGF-CX and/or FCTRX protein, mRNA, or genomic DNA in the postadministration sample or samples; and (vi) altering the administrationof the agent to the subject accordingly. For example, increasedadministration of the agent may be desirable to increase the expressionor activity of FGF-CX and/or FCTRX to higher levels than detected, i.e.,to increase the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of FGF-CX and/or FCTRX to lower levels than detected, i.e., todecrease the effectiveness of the agent.

Methods of Treatment

The invention provides for both prophylactic and therapeutic methods oftreating a subject at risk of (or susceptible to) a disorder or having adisorder associated with aberrant FGF-CX and/or FCTRX expression oractivity. The disorders include cardiomyopathy, atherosclerosis,hypertension, congenital heart defects, aortic stenosis, atrial septaldefect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus,pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD),valve diseases, tuberous sclerosis, scleroderma, obesity,transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia,prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer,fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenicpurpura, immunodeficiencies, graft versus host disease, AIDS, bronchialasthma, Crohn's disease; multiple sclerosis, treatment of AlbrightHereditary Ostoeodystrophy, and other diseases, disorders and conditionsof the like.

These methods of treatment will be discussed more fully, below.

Disease and Disorders

Diseases and disorders that are characterized by increased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with Therapeutics that antagonize(i.e., reduce or inhibit) activity. Therapeutics that antagonizeactivity may be administered in a therapeutic or prophylactic manner.Therapeutics that may be utilized include, but are not limited to: (i)an aforementioned peptide, or analogs, derivatives, fragments orhomologs thereof; (ii) antibodies to an aforementioned peptide; (iii)nucleic acids encoding an aforementioned peptide; (iv) administration ofantisense nucleic acid and nucleic acids that are “dysfunctional” (i.e.,due to a heterologous insertion within the coding sequences of codingsequences to an aforementioned peptide) that are utilized to “knockout”endoggenous function of an aforementioned peptide by homologousrecombination (see, e.g., Capecchi, 1989. Science 244:1288-1292); or (v)modulators (i.e., inhibitors, agonists and antagonists, includingadditional peptide mimetic of the invention or antibodies specific to apeptide of the invention) that alter the interaction between anaforementioned peptide and its binding partner.

Diseases and disorders that are characterized by decreased (relative toa subject not suffering from the disease or disorder) levels orbiological activity may be treated with Therapeutics that increase(i.e., are agonists to) activity. Therapeutics that upregulate activitymay be administered in a therapeutic or prophylactic manner.Therapeutics that may be utilized include, but are not limited to, anaforementioned peptide, or analogs, derivatives, fragments or homologsthereof; or an agonist that increases bioavailability.

Increased or decreased levels can be readily detected by quantifyingpeptide and/or RNA, by obtaining a patient tissue sample (e.g., frombiopsy tissue) and assaying it in vitro for RNA or peptide levels,structure and/or activity of the expressed peptides (or mRNAs of anaforementioned peptide). Methods that are well-known within the artinclude, but are not limited to, immunoassays (e.g., by Western blotanalysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/orhybridization assays to detect expression of mRNAs (e.g., Northernassays, dot blots, in situ hybridization, and the like).

Prophylactic Methods

In one aspect, the invention provides a method for preventing, in asubject, a disease or condition associated with an aberrant FGF-CXand/or FCTRX expression or activity, by administering to the subject anagent that modulates FGF-CX and/or FCTRX expression or at least oneFGF-CX and/or FCTRX activity. Subjects at risk for a disease that iscaused or contributed to by aberrant FGF-CX and/or FCTRX expression oractivity can be identified by, for example, any or a combination ofdiagnostic or prognostic assays as described herein. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the FGF-CX and/or FCTRX aberrancy, such that a diseaseor disorder is prevented or, alternatively, delayed in its progression.Depending upon the type of FGF-CX and/or FCTRX aberrancy, for example,an FGF-CX and/or FCTRX agonist or FGF-CX and/or FCTRX antagonist agentcan be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein. The prophylacticmethods of the invention are further discussed in the followingsubsections.

Therapeutic Methods

Another aspect of the invention pertains to methods of modulating FGF-CXand/or FCTRX expression or activity for therapeutic purposes. Themodulatory method of the invention involves contacting a cell with anagent that modulates one or more of the activities of FGF-CX and/orFCTRX protein activity associated with the cell. An agent that modulatesFGF-CX and/or FCTRX protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringcognate ligand of an FGF-CX and/or FCTRX protein, a peptide, an FGF-CXand/or FCTRX peptidomimetic, or other small molecule. In one embodiment,the agent stimulates one or more FGF-CX and/or FCTRX protein activity.Examples of such stimulatory agents include active FGF-CX and/or FCTRXprotein and a nucleic acid molecule encoding FGF-CX and/or FCTRX thathas been introduced into the cell. In another embodiment, the agentinhibits one or more FGF-CX and/or FCTRX protein activity. Examples ofsuch inhibitory agents include antisense FGF-CX and/or FCTRX nucleicacid molecules and anti-FGF-CX and/or FCTRX antibodies. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant expression or activity of an FGF-CX and/or FCTRX protein ornucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulates (e.g.,up-regulates or down-regulates) FGF-CX and/or FCTRX expression oractivity. In another embodiment, the method involves administering anFGF-CX and/or FCTRX protein or nucleic acid molecule as therapy tocompensate for reduced or aberrant FGF-CX and/or FCTRX expression oractivity.

Stimulation of FGF-CX and/or FCTRX activity is desirable in situationsin which FGF-CX and/or FCTRX is abnormally downregulated and/or in whichincreased FGF-CX and/or FCTRX activity is likely to have a beneficialeffect. One example of such a situation is where a subject has adisorder characterized by aberrant cell proliferation and/ordifferentiation (e.g., cancer or immune associated disorders). Anotherexample of such a situation is where the subject has a gestationaldisease (e.g., preclampsia).

Determination of the Biological Effect of the Therapeutic

In various embodiments of the invention, suitable in vitro or in vivoassays are performed to determine the effect of a specific Therapeuticand whether its administration is indicated for treatment of theaffected tissue.

In various specific embodiments, in vitro assays may be performed withrepresentative cells of the type(s) involved in the patient's disorder,to determine if a given Therapeutic exerts the desired effect upon thecell type(s). Compounds for use in therapy may be tested in suitableanimal model systems including, but not limited to rats, mice, chicken,cows, monkeys, rabbits, and the like, prior to testing in humansubjects. Similarly, for in vivo testing, any of the animal model systemknown in the art may be used prior to administration to human subjects.

Prophylactic and Therapeutic Uses of the Compositions of the Invention

The FGF-CX and/or FCTRX nucleic acids and proteins of the invention areuseful in potential prophylactic and therapeutic applications implicatedin a variety of disorders including, but not limited to: inflammatorybowel disease and disorders associated with FGF-CX, with FCTRX, or withboth FGF-CX and/or FCTRX.

As an example, a cDNA encoding the FGF-CX and/or FCTRX protein of theinvention may be useful in gene therapy, and the protein may be usefulwhen administered to a subject in need thereof. By way of non-limitingexample, the compositions of the invention will have efficacy fortreatment of patients suffering from: inflammatory conditions in thegastrointestinal tract, including but not limited to inflammatory boweldisease such as ulcerative colitis and Crohn's disease, growth andproliferative diseases such as cancer, angiogenesis, atheroscleroticplaques, collagen formation, cartilage and bone formation,cardiovascular and fibrotic diseases and diabetic ulcers. In addition,FCTRX nucleic acids and their encoded polypeptides will betherapeutically useful for the prevention of aneurysms and theacceleration of wound closure through gene therapy. Furthermore, FCTRXnucleic acids and their encoded polypeptides can be utilized tostimulate cellular growth, wound healing, neovascularization and tissuegrowth, and similar tissue regeneration needs. More specifically, aFCTRX nucleic acid or polypeptide may be useful in treatment of anemiaand leukopenia, intestinal tract sensitivity and baldness. Treatment ofsuch conditions may be indicated, e.g., in patients having undergoneradiation or chemotherapy, wherein treatment would minimize anyhyperproliferative side effects.

Both the novel nucleic acid encoding the FGF-CX and/or FCTRX protein,and the FGF-CX and/or FCTRX protein of the invention, or nucleic acid orprotein fragments, analogs, homologs or derivative thereof, may also beuseful in diagnostic applications, wherein the presence or amount of thenucleic acid or the protein are to be assessed. A further use could beas an anti-bacterial molecule (i.e., some peptides have been found topossess anti-bacterial properties). These materials are further usefulin the generation of antibodies which immunospecifically-bind to thenovel substances of the invention for use in therapeutic or diagnosticmethods.

EXAMPLES

It is shown in several Examples below that both FGF-CX and FCTRX inducethe growth and proliferation of various mammalian cells in culture. Itis further demonstrated in animal models of inflammatory bowel diseasethat these proteins have beneficial effects in treating, amelioratingand delaying the onset of inflammatory bowel disease. By “treating” ismeant the administration of a protein used in the present invention to asubject suffering from a pathology such as inflammatory bowel diseasewith the objective of providing a beneficial therapeutic effect. By“ameliorating” a pathology such as inflammatory bowel disease, it ismeant that a) in a subject in which the pathology is becoming moresevere, one or more symptoms of the pathology cease becoming more severeand stabilize or improve; or b) in a subject in which the pathology isconsidered to be at a stable state, one or more symptoms of thepathology improve or become less severe. By “delaying the onset” of apathology such as inflammatory bowel disease, it is meant thatadministering a prophylactic dose or dosing regimen of a therapeuticagent such as the FGF-CX and FCTRX proteins employed in the presentinvention results in the delay of appearance, or the delay of worsening,of one or more symptoms of a pathology such as inflammatory boweldisease. Such a delay may be for an indeterminate period, in which thesymptoms essentially never appear or never worsen, or it may be for Amore limited period, in which the symptoms appear or worsen at a latertime than would be expected, based on the experience of patients nottreated by the compositions envisioned in the present methods, in theabsence of administering the therapeutic agent.

The results of experiments reported below in three Examples indicatethat, in mice in which inflammatory bowel disease is induced by oraladministration of DSS for 7 days, simultaneous treatment with the growthfactors employed here during the course of exposure to DSS lead tosignificant therapeutic benefits compared to untreated DSS controls.

An additional Example reports results on rats treated with indomethacinwhich results in gross and histopathologic intestinal alterations thatare similar to those occurring in Crohn's Disease. Administration ofCG53135 (0.2 mg/kg iv) to indomethacin-treated rats results insignificant reductions in weight loss, small intestine weight, absoluteneutrophil counts, and jejunal necrosis and inflammation scores.

Example 1 Identification of the FGF-CX Gene

The FGF-CX gene was identified following a TBLASTN (Altschul, S. F.,Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) J. Mol. Biol.215, 403-410) search of Genbank human genomic DNA sequences with XenopusFGF-CX (Koga, C., Adati, N., Nakata, K., Mikoshiba, K., Furuhata, Y.,Sata, S., Tei, H., Sakati, Y., Kurokawa, T., Shiokawa, K. & Yokoyama, K.K. (1999) Biochem. Biophys. Res. Comm. 261, 756-765; Accession No.AB012615) as query. This search identified a locus (Accession No.AB020858) of high homology on chromosome 8. Intron/exon boundaries werededuced using standard consensus splicing parameters (Mount, S. M.(1996) Science 271, 1690-1692), together with homologies derived fromknown FGFs. The FGF-CX initiation codon localizes to bp 16214 of thesequence of AB020858, and the remaining 3′ portion of this exoncontinues to bp 15930. The 5′ UTR of FGF-CX was extended upstream of theinitiation codon by an additional 606 bp using public ESTs (AccessionNos. AA232729, AA236522, AI272876 and AI272878). The remaining structureof the FGF-CX gene as it relates to locus AB020858 is as follows: intron1 (bp 15929-9942); exon 2 (bp 9941-9838); intron 2 (bp 9837-7500); exon3 (begins at bp 7499).

The gene discovered by the procedure in the preceding paragraph includes3 exons and 2 introns. The DNA sequence predicts an ORF of 211 aminoacid residues (see Table 1), with an in-frame stop codon 117 bp upstreamof the initiator methionine. The DNA segment from which the gene wasmined maps to chromosome 8p21.3-p22, a location that was confirmed byradiation hybrid analysis.

Example 2 Molecular Cloning of the Sequence Encoding a FGF-CX Protein

Oligonucleotide primers were designed for the amplification by PCR of aDNA segment, representing an open reading frame, coding for the fulllength FGF-CX. The forward primer includes a BglII restriction site(AGATCT) and a consensus Kozak sequence (CCACC). The reverse primercontains an in-frame XhoI restriction site for further subcloningpurposes. Both the forward and the reverse primers contain a 5′ clampsequence (CTCGTC). The sequences of the primers are the following:

FGF-CX-Forward: 5′-CTCGTC AGATCT CCACC ATG GCT CCC (SEQ ID NO:15) TTAGCC GAA GTC-3′ FGF-CX-Reverse: 5′-CTCGTC CTCGAG AGT GTA CAT CAG TAG (SEQID NO:16) GTC CTT G-3′

PCR reactions were performed using a total of 5ng human prostate cDNAtemplate, 1 μM of each of the FGF-CX-Forward and FGF-CX-Reverse primers,5 micromoles dNTP (Clontech Laboratories, Palo Alto Calif.) and 1microliter of 50xAdvantage-HF 2 polymerase (Clontech Laboratories) in 50microliter volume. The following PCR reaction conditions were used:

a) 96° C. 3 minutes b) 96° C. 30 seconds denaturation c) 70° C. 30seconds, primer annealing. This temperature was gradually decreased by1° C./cycle. d) 72° C. 1 minute extension. Repeat steps (b)-(d) tentimes e) 96° C. 30 seconds denaturation f) 60° C. 30 seconds annealingg) 72° C. 1 minute extension Repeat steps (e)-(g) 25 times h) 72° C. 5minutes final extension

A single PCR product, with the expected size of approximately 640 bp,was isolated after electrophoresis on agarose gel and ligated into apCR2.1 vector (Invitrogen, Carlsbad, Calif.). The cloned insert wassequenced using vector specific M13 Forward(−40) and M13 Reverseprimers, which verified that the nucleotide sequence was 100% identicalto the sequence in Table 1 (SEQ ID NO:1) inserted directly between theupstream BglII cloning site and the downstream XhoI cloning site. Thecloned sequence constitutes an open reading frame coding for thepredicted FGF-CX full length protein. The clone is calledTA-AB02085-S274-F19.

Example 3 Preparation of Mammalian Expression Vector pCEP4/Sec

The oligonucleotide primers pSec-V5-His Forward CTCGT CCTCG AGGGT AAGCCTATCC CTAAC (SEQ ID NO:17) and pSec-V5-His Reverse CTCGT CGGGC CCCTGATCAG CGGGT TTAAA C (SEQ ID NO:18), were designed to amplify a fragmentfrom the pcDNA3.1-V5His (Invitrogen, Carlsbad, Calif.) expression vectorthat includes V5 and His6. The PCR product was digested with XhoI andApal and ligated into the XhoI/ApaI digested pSecTag2 B vector harboringan Ig kappa leader sequence (Invitrogen, Carlsbad Calif.). The correctstructure of the resulting vector, pSecV5His, including an in-frameIg-kappa leader and V5-His6 was verified by DNA sequence analysis. Thevector pSecV5His was digested with PmeI and NheI to provide a fragmentretaining the above elements in the correct frame. The PmeI-NheIfragment was ligated into the BamHI/Klenow and NheI treated vector pCEP4(Invitrogen, Carlsbad, Calif.). The resulting vector was named pCEP4/Secand includes an in-frame Ig kappa leader, a site for insertion of aclone of interest, and the V5 epitope and 6xHis under control of thePCMV and/or the PT7 promoter. pCEP4/Sec is an expression vector thatallows heterologous protein expression and secretion by fusing anyprotein into a multiple cloning site following the Ig kappa chain signalpeptide. Detection and purification of the expressed protein are aidedby the presence of the V5 epitope tag and 6xHis tag at the C-terminus(Invitrogen, Carlsbad, Calif.).

Example 4 Expression of FGF-CX in Human Embryonic Kidney (HEK) 293 Cells

The BglII-XhoI fragment containing the FGF-CX sequence was isolated fromTA-AB02085-S274-F19 (Example 2) and subcloned into the BamHI-XhoIdigested pCEP4/Sec to generate the expression vector pCEP4/Sec-FGF-CX.The pCEP4/Sec-FGF-CX vector was transfected into 293 cells using theLipofectaminePlus reagent following the manufacturer's instructions(Gibco/BRL/Life Technologies, Rockville, Md.). The cell pellet andsupernatant were harvested 72 hours after transfection and examined forFGF-CX expression by Western blotting (reducing conditions) with ananti-V5 antibody. FIG. 1 shows that FGF-CX is expressed as a polypeptidehaving an apparent molecular weight (Mr) of approximately 34 kDaproteins secreted by 293 cells. In addition a minor band is observed atabout 31 kDa.

Example 5 Expression of FGF-CX in E. coli

The vector pRSETA (In Vitrogen Inc., Carlsbad, Calif.) was digested withXhoI and NcoI restriction enzymes. Oligonucleotide linkers of thesequence

-   -   5′ CATGGTCAGCCTAC 3′ (SEQ ID NO:19) and    -   5′ TCGAGTAGGCTGAC 3′ (SEQ ID NO:20)

were annealed at 37 degree Celsius and ligated into the XhoI-NcoItreated pRSETA. The resulting vector was confirmed by restrictionanalysis and sequencing and was named pETMY. The BgllI-XhoI fragment ofthe sequence encoding FGF-CX (see Example 2) was ligated into vectorpETMY that was digested with BamHI and XhoI restriction enzymes. Theexpression vector is named pETMY-FGF-CX. In this vector, hFGF-CX wasfused to the 6xHis tag and T7 epitope at its N-terminus. The plasmidpETMY-FGF-CX was then transfected into the E. coli expression hostBL21(DE3, pLys) (Novagen, Madison, Wis.) and expression of proteinFGF-CX was induced according to the manufacturer's instructions. Afterinduction, total cells were harvested, and proteins were analyzed byWestern blotting using anti-HisGly antibody (Invitrogen, Carlsbad,Calif.). FIG. 2 shows that FGF-CX was expressed as a protein of apparentmolecular weight Mr approximately 32 kDa.

Example 6 Comparison of Expression of Recombinant FGF-CX Protein Withand Without a Cloned Signal Peptide

a) Expression Without a Signal Peptide

As noted in the Detailed Description of the Invention, FGF-CX apparentlylacks a classical amino-terminal signal sequence. To determine whetherFGF-CX is secreted from mammalian cells, cDNA obtained as the BglII-XhoIfragment, encoding the full length FGF-CX protein, was subcloned fromTA-AB02085-S274-F19 (Example 2) into BamHI/XhoI-digested pcDNA3.1(Invitrogen). This provided a mammalian expression vector designatedpFGF-CX. This construct incorporates the V5 epitope tag and apolyhistidine tag into the carboxy-terminus of the protein to aid in itsidentification and purification, respectively, and should generate apolypeptide of about 27 kDa. Following transient transfection into 293human embryonic kidney cells, conditioned media was harvested 48 hr posttransfection.

In addition to secretion of FGF-CX into conditioned media, it also foundto be associated with the cell pellet/ECM (data not shown). Since FGFsare known to bind to heparin sulfate proteoglycan (HSPG) present on thesurface of cells and in the extracellular matrix (ECM), the inventorsinvestigated the possibility that FGF-CX was sequestered in this manner.To this end, FGF-CX-transfected cells were extracted by treatment with0.5 ml DMEM containing 100 μM suramin, a compound known to disrupt lowaffinity interactions between growth factors and HSPGs (La Rocca, R. V.,Stein, C. A. & Myers, C. E. (1990) Cancer Cells 2, 106-115), for 30 minat 4° C. The suramin-extracted conditioned media was then harvested andclarified by centrifigation (5 min; 2000×g).

The conditioned media and the suramin extract were then mixed with equalvolumes of 2×gel-loading buffer. Samples were boiled for 10 min,resolved by SDS-PAGE on 4-20% gradient polyacrylamide gels (Novex, DanDiego, Calif.) under reducing conditions, and transferred tonitrocelluose filters (Novex). Western analysis was performed accordingto standard procedures using HRP-conjugated anti-V5 antibody(Invitrogen) and the ECL detection system (Amersham Pharmacia Biotech,Piscataway, N.J.).

One band having the expected Mr was identified in conditioned media from293 cells transfected with pFGF-CX (FIG. 3, Panel A, lane 1).Conditioned media from cells transfected with control vector did notreact with the antibody (FIG. 3, Panel A, lane 5). After suramintreatment, it was found that a significant quantity of FGF-CX could infact be released from the cell surface/ECM, indicating that HSPGs arelikely to play a role in sequestering this protein (FIG. 3, Panel A,lane 2). These results indicate that FGF-CX can be secreted without aclassical signal peptide.

Recombinant FGF-CX protein stimulates DNA synthesis and cellproliferation, effects that are likely to be mediated via high affinitybinding of FGF-CX to a cell surface receptor, and modulated via lowaffinity interactions with HSPGs. The suramin extraction data suggeststhat FGF-CX binds to HSPGs present on the cell surface and/or the ECM.

b) Expression With a Signal Peptide

With the goal of enhancing protein secretion, a construct(pCEP4/Sec-FGF-CX) was generated in which the FGF-CX cDNA was fused inframe with a cleavable amino-terminal secretory signal sequence derivedfrom the Igκ gene. The resulting protein also contained carboxy-terminalV5 and polyhistidine tags as described above for pFGF-CX. Followingtransfection into 293 cells, a protein product having the expected Mr ofabout 31 kDa was obtained, and suramin was again found to release asignificant quantity of sequestered FGF-CX protein (FIG. 3, Panel A;lanes 3 and 4). As expected, pCEP4/Sec-FGF-CX generated more solubleFGF-CX protein than did pFGF-CX.

Results similar to those described above for 293 cells were alsoobtained with NIH 3T3 cells (FIG. 3, Panel B).

Example 7 Expression of FGF-CX

FGF-CX was expressed essentially as described in Example 5. The proteinwas purified using Ni²⁺-affinity chromatography, subjected to SDS-PAGEunder both reducing and nonreducing conditions, and stained usingCoomassie Blue. The results are shown in FIG. 4. It is seen that underboth sets of conditions, the protein migrates with an apparent molecularweight of approximately 29-30 kDa.

Example 8 Stimulation of Bromodeoxyuridine Incorporation by RecombinantFGF-CX

A dose response experiment for incorporation of BrdU was carried outusing human renal carcinoma cells (786-0; American Type CultureCollection, Manassas, Va.). 293-EBNA cells (Invitrogen) were transfectedusing Lipofectamine 2000 according to the manufacturer's protocol (LifeTechnologies, Gaithersburg, Md.). Cells were supplemented with 10% fetalbovine serum (FBS; Life Technologies) 5 hr post-transfection. Togenerate protein for BrdU and growth assays (Example 10), cells werewashed and fed with Dulbecco's modified Eagle medium (DMEM; LifeTechnologies) 18 hr post-transfection. After 48 hr, the media wasdiscarded and the cell monolayer was incubated with 100 μM suramin(Sigma, St. Louis, Mo.) in 0.5 ml DMEM for 30 min at 4° C. Thesuramin-extracted conditioned media was then removed, clarified bycentrifugation (5 min; 2000×g), and subjected to TALON metal affinitychromatography according to the manufacturer's instructions (Clontech,Palo Alto, Calif.) taking advantage of the carboxy-terminalpolyhistidine tag. Retained fusion protein was released by washing thecolumn with imidazole.

To generate control protein, 293-EBNA cells were transfected with pCEP4plasmid (Invitrogen) and subjected to the purification procedureoutlined above.

Recombinant FGF-CX was tested for its ability to induce DNA synthesis ina bromodeoxyuridine (BrdU) incorporation assay. 786-0 cells werecultured in 96-well plates to approximately 100% confluence, washed withDMEM, and serum-starved in DMEM for 24 hr. Recombinant FGF-CX or controlprotein was then added to the cells for 18 hr. The BrdU assay wasperformed according to the manufacturer's specifications (RocheMolecular Biochemicals, Indianapolis, Ind.) using a 5 hr BrdUincorporation time.

The results are shown in FIG. 5, in which FGF-CX is designated “20858”.It is seen that FGF-CX stimulates proliferation of renal carcinoma cellsby more than 4-fold over controls, with a half-effective dose beingabout 2.5 ng/mL.

Example 9 Receptor Binding Specificity of FGF-CX

To determine the receptor binding specificity of FGF-CX, we examined theeffect of soluble FGF receptors (FGFRs) on the induction of DNAsynthesis in NIH 3T3 cells by recombinant FGF-CX. Four receptors havebeen identified to date (Klint P and Claesson-Welsh L. Front. Biosci.,4: 165-177, 1999; Xu X, et al. Cell Tissue Res., 296: 33-43, 1999).Soluble receptors for FGFR1β(IIIc), FGFR2α(IIIb), FGFR2β(IIIb),FGFR2α(IIIc), FGFR3α (IIIc) and FGFR4 were utilized. It was found thatsoluble forms of each of these FGFRs were able to specifically inhibitthe biological activity of FGF-CX (see FIG. 6). Complete or nearlycomplete inhibition was obtained with soluble FGFR2α(IIIb),FGFR2β(IIIb), FGFR2α(IIIc), and FGFR3α(IIIc), whereas partial inhibitionwas achieved with soluble FGFR1β(IIIc) and FGFR4. None of the solublereceptor reagents interfered with the induction of DNA synthesis byPDGF-BB, thereby demonstrating their specificity. The integrity of eachsoluble receptor reagent was demonstrated by showing its ability toinhibit the induction of DNA synthesis by aFGF (acidic FGF), a factorknown to interact with all of the FGFRs under analysis.

Example 10 Cloning and Expression of an N-terminal Deletion Form ofFGF-CX

E. coli strain BL21 (DE3) (Invitrogen) harboring the plasmid pET24a-FGF20X-del54-codon were grown in LB medium at 37° C. This plasmidencodes the C-terminal portion of FGF-CX beginning at position 55. Whencell densities reached an OD of 0.6, IPTG was added to finalconcentration of 1 mM. Induced cultures were then incubated for anadditional 4 hours at 37° C. Cells were harvested by centrifugation at3000×g for 15 minutes at 4° C., suspended in PBS and then disrupted withtwo passes through a microfluidizer. To separate soluble and insolubleproteins, the lysate was subjected to centrifugation at 10,000×g for 20minutes at 4° C. The insoluble fraction (pellet) was extracted with PBScontaining 1M L-arginine. The remaining insoluble material was thenremoved by centrifugation and the soluble fraction of the arginineextract was filtered through 0.2 micron low-protein binding membrane andanalyzed by SDS PAGE. The result is shown in FIG. 7, which indicatesthat the product is a polypeptide with an apparent molecular weight ofapproximately 20 kDa (see arrow). N-terminal sequencing of the expressedpolypeptide provides the sequence AQLAHLHGILRRRQL which is 100%identical to residues 54-64 of FGF-CX (Table 1, SEQ ID NO:2).

Example 11 Stimulation of Bromodeoxyuridine Incorporation into NIH 3T3Cells in Response to a Truncated Form of FGF-CX

A vector expressing residues 24-211 of FGF-CX, referred to as(d1-23)FGF-CX, was prepared. See Table 1 and SEQ ID NO:2. Theincorporation of BrdU by NIH 3T3 cells treated with conditioned mediumobtained using the vector incorporating this truncated form was comparedto the incorporation in response to treatment with conditioned mediumusing a vector encoding full length FGF-CX. This experiment was carriedout as described in Example 8.

The results are shown in FIG. 8. It is seen that (d1-23)FGF-CX retainshigh activity at the lowest concentration tested, 10 ng/mL. At thisconcentration, the activity of full length FGF-CX has fallenconsiderably, approaching the level of the control. It is estimated that(d1-23)FGF-CX may be at least 5-fold more active than full lengthFGF-CX.

Example 12 Molecular Cloning of a Mature FCTR1 Form (30664188.0.m99)Polypeptide from Cline 30664188.0.99

A mature form of clone 30664188.0.99, coding for residues 24 to 370 ofthe amino acid sequence of Table 2 (SEQ ID NO:4) was cloned. Thisfragment was designated 30664188.0.m99 and corresponds to thepolypeptide sequence remaining after a signal peptide predicted to becleaved between residues 23 and 24 has been removed. The followingoligonucleotide primers were designed to PCR amplify the predictedmature form of 30664188.0.99: 30664188 Eco Forward—CTCGTC GAATTC ACC CCGCAG AGC GCA TCC ATC AAA GC (SEQ ID NO:21), and 3066418 XhoReverse—CTCGTC CTC GAG TCG AGG TGG TCT TGA GCT GCA GAT ACA (SEQ IDNO:22).

The forward primer included an in frame EcoRI restriction site, and thereverse primer included an XhoI restriction site. The EcoRI/Xholfragment is compatible with the pET28a E. coli expression vector andwith the pMelV5His baculovirus expression vector.

PCR reactions were set up using 5 ng human spleen and fetal lung cDNAtemplates. The reaction mixtures contained 1 microM of each of the30664188 Eco Forward and 30664188 Xho Reverse primers, 5 micromoles dNTP(Clontech Laboratories, Palo Alto Calif.) and 1 microliter of50xAdvantage-HF 2 polymerase (Clontech Laboratories, Palo Alto Calif.)in 50 microliter volume. The following reaction conditions were used:

a) 96° C. 3 minutes b) 96° C. 30 seconds denaturation c) 70° C. 30seconds, primer annealing. This temperature was gradually decreased by1° C. per cycle d) 72° C. 1 minute extension. Repeat steps b-d 10 timese) 96° C. 30 seconds denaturation f) 60° C. 30 seconds annealing g) 72°C. 1 minute extension Repeat steps e-g 25 times h) 72° C. 5 minutesfinal extension

The amplified product expected to have 1041 bp was detected by agarosegel electrophoresis in both samples. The fragments were purified fromagarose gel and ligated to pCR2.1 vector (Invitrogen, Carlsbad, Calif.).The cloned inserts were sequenced using M13 Forward, M13 Reverse and thefollowing gene specific primers:

3066418 S1: GGA CGA TGG TGT GGA CAC AAG, (SEQ ID NO:23) 3066418 S2: CTTGTG TCC ACA CCA TCG TCC, (SEQ ID NO:24) 3066418 S3: TAT CGA GGC AGG TCATAC CAT and (SEQ ID NO:25) 3066418 S4: ATG GTA TGA CCT GCC TCG ATA. (SEQID NO:26)

The cloned inserts were verified as an open reading frame coding for thepredicted mature form of 30664188.0.99. The construct derived from fetallung, called 30664188-S311a, was used for further subcloning intoexpression vectors (see below). The nucleotide sequence of 30664188-S11awithin the restriction sites was found to be 100% identical to thecorresponding fragment in the ORF of 30664188.0.99 (Table 2; SEQ IDNO:4).

Example 13 Expression of 30664188.m99 Polypeptide in E. coli

The vector pRSETA (InVitrogen Inc., Carlsbad, Calif.) was digested withXhoI and NcoI restriction enzymes. Oligonucleotide linkersCATGGTCAGCCTAC (SEQ ID NO:27); and TCGAGTAGGCTGAC (SEQ ID NO:28) wereannealed at 37 degrees Celsius and ligated into the XhoI-NcoI treatedpRSETA. The resulting vector was confirmed by restriction analysis andsequencing and was named pETMY. The BamHI-XhoI fragment containing the30664188 sequence (Example 12) was ligated into BamHI-XhoI digestedpETMY. The resulting expression vector was named pETMY-30664188. In thisvector, 30664188 is fused to the T7 epitope and a 6xHis tag at itsN-terminus The plasmid pETMY-30664188 was then transfected into the E.coli expression host BL21(DE3, pLys) (Novagen, Madison, Wis.) andexpression of the protein was induced according to the manufacturer'sinstructions. After induction, the E. coli cells were harvested, andproteins were analyzed by Western blotting using anti-His6Gly antibody(Invitrogen, Carlsbad, Calif.). FIG. 9 shows that the resultingpolypeptide, termed 30664188.m99 herein, was expressed as a protein ofapparent molecular weight 40 kDa. This approximates the molecular weightexpected for the 30664188.m99 sequence.

Example 14 Expression of 30664188.m99 Polypeptide in Human EmbryonicKidney 293 Cells

The EcoRI-XhoI fragment containing the 30664188.m99 sequence wasisolated from 30664188-S311a (Example 12) and subcloned into the vectorpE28a (Novagen, Madison, Wis.) to give the plasmid pET28a-30664188.Subsequently, pET28a-30664188 was partially digested with BamHIrestriction enzyme, and then completely digested with XhoI. A fragmentof 1.1 kb was isolated and ligated into BamHI-XhoI digested pCEP4/Sec(Example 3) to generate expression vector pCEP4/Sec-30664188.m99. ThepCEP4/Sec-30664188.m99. vector was transfected into human embryonickidney 293 cells (ATCC No. CRL-1573, Manassas, Va.) using theLipofectaminePlus reagent following the manufacturer's instructions(Gibco/BRL/Life Technologies, Rockville, Md.). The cell pellet andsupernatant were harvested 72 hours after transfection and examined forexpression of the 30664188.m99 protein by Western blotting of anSDS-PAGE run under reducing conditions using an anti-V5 antibody. FIG.10 shows that 30664188.m99 is expressed as three discrete protein bandsof apparent molecular weight 50, 60, and 98 kDa secreted by 293 cells.The 50 kDa band migrated at a sized expected for a monomer glycosylatedform of 30664188.m99, and the 98 kDa band migrated at a size consistentwith a dimer of the monomer form.

Example 15 Expression and Purification of 30664188.m99 Protein

HEK 293 cells were grown in Dulbecco's modified eagle's medium(DMEM)/10% fetal bovine serum medium to 90% confluence. The cells weretransfected with pCEP4sec or pCEP4sec/30664188.m99 using Lipofectamine2000 according to the manufacturer's specifications (Gibco/BRL/LifeTechnologies, Rockville, Md.). Transfected cells were incubated for 2days with DMEM and conditioned medium was prepared by collection of cellsupernatants. The conditioned medium was enriched by Talon metalaffinity chromatography (Clontech, Palo Alto, Calif.). Briefly, 7 ml ofconditioned medium was incubated with 1 ml of Talon metal affinity resinin spin columns. The spin columns were washed twice with one ml of PBS.The columns were then eluted twice with 0.65 ml of PBS/0.5M imidazole pH8.0 and the eluates pooled. Imidazole was removed by buffer exchangedialysis into PBS using Microcon centrifugal filter devices (MilliporeCorp., Bedford, Mass.). The enriched gene products were stored at 4° C.

The purified protein obtained was subjected to SDS-PAGE under reducingconditions and probed with an anti-V5 antibody, which was detected withan enzyme label. The results of two separate transfection andpurification runs are shown in the gels. They show that the product is amixture of V5-containing polypeptides. The largest has an apparentmolecular weight of about 50 kDa (FIG. 11, Panel B). The program ProSitepredicts one N-glycosylation site in the mature protein. Glycosylationmay explain the apparent molecular weight found. Thus the 50 kDa band isconsistent with the length expected for full length gene product. Otherbands, preponderantly having apparent molecular weights of about 20-25kDa also arise. These are presumed to be the result of proteolysisoccurring either intracellularly within the 293 cells or extracellularlyafter secretion from them. In another run (not shown) the broad bandextending from about 6 kDa to about 14 kDa is reolved into two bands ofabout 7-8 kDa and about 10 kDa.

Example 16 The Clone 30664188.0.m99 Protein Induces Cellular DNASynthesis

Human CCD-1070 fibroblast cells (ATCC No. CRL-2091, Manassas, Va.) ormurine NIH 3T3 (ATCC No. CRL-1658, Manassas, Va.) fibroblast cells werecultured in DMEM supplemented with 10% fetal bovine serum or 10% calfserum respectively. Fibroblasts were grown to confluence at 37° C. in10% CO₂/air. Cells were then starved in DMEM for 24 h. pCEP4/Sec(Example 3) or pCEP4/Sec/30664188.m99 (Example 14) enriched conditionedmedium was added (10 microL/100 microL of culture ) for 18 h. BrdU (10uM) was then added and incubated with the cells for 5 h. BrdUincorporation was assayed by colorimetric immunoassay according to themanufacturer's specifications (Boehringer Mannheim, Indianapolis, Ind.).

FIG. 12 demonstrates that 30664188.m99 induced an approximate four- tofive-fold increase in BrdU incorporation in either cell type compared tocells treated with control conditioned medium or untreated cells. Theproliferative increase observed was similar to the increase in BrdUincorporation induced by platelet derived FCTRX (PDGF), basic fibroblastgrowth factor (bFGF), or serum treatment. Additionally, 30664188.m99partially purified conditioned medium did not induce BrdU incorporationin human MG-63 epithelial cells or CCD1106 keratinocytes (data notshown). These results suggest that 30664188 selectively induces DNAsynthesis in human and mouse fibroblasts, but not in epithelial celllines.

In separate experiments, CCD-1070 cells and MG-63 osteosarcoma cells(ATCC Cat. No. CRL-1427) treated with pCEP4/Sec/30664188 eachincorporated BrdU in a dose-dependent fashion, with 1 ug/mL providingthe full effect (approximately 2.5- to 3-fold increase over control),100 ng/mL providing slightly less than one-half the effect, and 10 and 1ng/mL providing approximately control levels of incorporation.Furthermore, the dose response of NIH 3T3 cells shows that a 50%response occurs between doses of 10 and 50 ng/mL ofpCEP4/Sec/30664188.M99 (FIG. 13).

In additional dose titration experiments using both NIH/3T3 cells andCCD 1070 cells, the half maximal effect occurred at or below 25 ng/mL.

Example 17 Induction of Proliferation of NIH 3T3 Cells by 30664188.m99

Murine NIH 3T3 fibroblasts were plated at 40% confluency and cultured inDMEM supplemented with 10% fetal bovine serum or 10% calf serum for 24hrs. The culture medium was removed and replaced with an equivalentvolume of pCEP4/Sec (Example 3) or pCEP4/Sec/30664188.m99.m99. (Example14) conditioned medium. After 48 h, cells were photographed with a ZeissAxiovert 100. Cell numbers were determined by trypsinization followed bycounting using a Coulter Z1 Particle Counter.

Treatment of NIH 3T3 fibroblasts with conditioned medium from 30664188transfected HEK293 kidney epithelial cells resulted in a 6 to 8 foldincrease in cell number over a two day period (FIG. 14). Cells treatedwith control conditioned medium from HEK293 cells transfected with thepCEP4/Sec vector alone demonstrated little or no growth (FIG. 14).

To determine whether 30664188.m99 conditioned medium was able to inducephenotypic changes characteristic of cellular transformation, cellstreated with either 30664188 conditioned medium or mock conditionedmedium were examined by light microscopy. FIG. 15 shows that NIH 3T3cells treated with 30664188.m99, but not control treated NIH 3T3 cells,showed a marked increase in cell number, as well as refractileproperties. Loss of contact inhibition of growth was evident. Thecobblestone appearance characteristic of confluent NIH 3T3 cells waslost and density independent growth was evident. The latter was alsosuggested by the more rounded appearance of the NIH 3T3 cells due tosubtle retraction. Transfection of pCEP4/Sec/30664188.m99.m99 alsoshowed nearly identical potency in transformation potential 2 to 5 daysin culture. After 7 to 10 days in culture, however, the morphologicallytransformed phenotype appeared to revert.

Example 18 Induction of Proliferation of Human Primary Osteoblast Cellsby the 30664188 Protein

In an experiment similar to that described in Example 17, human primaryosteoblast cells (NHost; Clonetics)also underwent a dose-dependentincrease in cell number by 3- to 4-fold (FIG. 16). The dose required toelicit a 50% response in FIG. 16 is below 100 ng/mL ofpCEP4/Sec/30664188.m99. In addition, Jurkat cells contacted withpartially purified conditioned medium containing the 30664188 geneproduct exhibited a doubling of BrdU uptake compared to the medium frommock transfection, whereas the same cells contacted with 13 otherCuraGen Corporation gene products thought to have growth promotingactivity elicited no effect.

In summary, the observations that the 30664188 protein induces DNAsynthesis (Example 16), cell growth (Examples 16 and 17), andmorphological transformation (Example 17) indicate that the 30664188protein possesses growth promoting and stimulating properties.

Example 19 Purification of Intact and Cleaved Products of the30664188.m99 Protein

It was observed that in certain experiments treatment with the vectorpCEP4/Sec/30664188.m99 did not result in DNA synthesis or cellproliferation. In additional experiments, medium conditioned with30664188.m99 was obtained from HEK 293 cells grown in the presence ofserum (Examples 15-17). The 30664188.m99 gene product was purified bycation exchange chromatography, followed by nickel affinitychromatography. The protein product was run under nonreducing andreducing conditions on SDS-PAGE, and developed by Coomassie stain. Theresults are shown in FIG. 17, Panels A and B. In the presence of serum,the 30664188.m99 gene product appeared as a protein of about 35 kDaunder nonreducing conditions (FIG. 17 Panel B). However, thispolypeptide appears as three degraded bands when run under reducingconditions. The apparent molecular weights of the two bands were 22-25kDa (band I), about 16 kDa (band II) and about 5-6 kDa (band III).N-terminal amino acid analysis of these fragments indicates that bands Iand II both appear to result from cleavage between residues 247 and 248,such that the peptide product begins at residue 248 of the 30664188.0.99(Table 2, SEQ ID NO:4) amino acid sequence, and that band III begins atresidue 339. These results are consistent with cleavage of thepolypeptide corresponding to band I to provide the fragments of bands IIand III. It is possible that the 35 kDa band observed under nonreducingconditions is a dimer composed of band I, and/or the bonded polypeptidecomposed of bands II and III, observed under reducing conditions.

Amino terminal analysis indicates that the gene product frompCEP4sec/30664188.m99-transfected 293 cells grown in the presence ofserum, isolated according to the procedure described above, is acarboxyl-terminal fragment of the full length protein. The 35 kDa bandfound under nonreducing conditions is termed p35 below. These resultsare expanded in Example 21.

When 293 cells were cultured in the absence of serum, and the sameisolation and detection procedure described in the preceding paragraphis followed, a different gene product is observed. Under nonreducingconditions a band was found at about 85 kDa (FIG. 17 Panel A). Thisprotein is termed p85 below. The corresponding gene product observedunder reducing conditions a major band is found at about 53-54 kDa.N-terminal amino acid analysis of this gene product provides the aminoacids at the multiple cloning site used in pCEP4sec/30664188.m99(Example 14). The residues corresponding to the Ig kappa leadersequence, cloned upstream from the multiple cloning site, are absent.These results indicate that the gene product obtained in the absence ofserum represents the full amino acid sequence encoded inpCEP4sec/30664188.m99. The p85 polypeptide is thought to be a dimer ofthe 50 kDa species observed on reducing SDS-PAGE. These results areexpanded in Example 21.

Example 20 Activity of Intact and Cleaved Fragments of the 30664188.m99Protein

Purified p85 and p35 FCTRX proteins were separately applied to NIH 3T3cells in a range of concentrations. Incorporation of BrdU was evaluatedas described in Example 8. The results are shown in FIG. 18. It is seenthat p85 has growth-promoting activity that does not differ from controllevels except at the highest concentration used (bars 4-10). p35, on theother hand, was at least as active, if not more so, than unfractionatedpCEP4/Sec/30664188.m99 conditioned medium (bars 11-17). Theconcentration of p35 giving 50% of the maximum DNA synthesis fallsbetween 20 and 50 ng/mL.

These results suggest that the p35 fragment derived from intact30664188.m99 has growth-promoting activity but that the intact dimericform of the .m99 protein, p85, does not.

Example 21 Purification of Recombinant PDGF DD

The gene product of PDGFD was expressed in HEK293 cells grown on porousmicrocarriers (Cultisphere-GL, Hyclone; Logan, Utah) in 1 L spinnerflasks. As noted in Examples 2 and 4, the recombinant PDGF D geneincludes a 6xHis fusion at the 3′ end. Cells were grown in DMEM/F12media containing 1% penicillin/streptomycin in the presence or absenceof 5% fetal bovine serum (FBS). The conditioned medium was harvested bycentrifugation (4000×g for 15 minutes at 4° C.) and loaded onto a POROSHS50 column (PE Biosystems; Foster City, Calif.), pre-equilibrated with20 mM Tris-acetate (pH 7.0). After washing with the equilibrationbuffer, bound proteins were eluted with a NaCl step gradient (0.25 M,0.5 M, 1.0 M and 2.0 M). Fractions containing PDGF DD p35 (1.0 M NaClstep elution) or p85 (0.5 M NaCl step elution) (see Example 19) werepooled and diluted with an equal volume of phosphate-buffered saline(PBS), pH 8.0 containing 0.5 M NaCl, then loaded onto a POROS MC20column pre-charged with nickel sulfate (PE Biosystems). After washingwith PBS/0.5 M NaCl, bound proteins were eluted with a linear gradientof imidazole (0-0.5 M). Fractions containing PDGF DD (homodimers ofPDGFD) (100-150 mM imidazole) were pooled and dialyzed twice against1000 volumes of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl. The protein puritywas estimated to be >95% by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE; 4-20% Tris-glycine gradient gel; Invitrogen,Carlsbad, Calif.) analysis (See, for example, the results in Example 19,including FIG. 17).

Biochemical Properties of PDGF D. To examine the biochemical propertiesof the gene product of PDGF D, the cDNA encoding PDGF D protein wassubcloned into a mammalian expression vector, pCEP4/Sec-30664188m99(Example 14). This construct incorporates an epitope tag (V5) and apolyhistidine tag into the COOH terminus of the protein to aid in itsidentification and purification (expression vectorpCEP4/Sec-30664188m99; Example 14).

Following transfection into 293 HEK cells and growth in serum-freeculture, a secreted polypeptide with an apparent molecular weight of ˜49kDa (p49 species) was identified by Western blot analysis under reducingconditions (FIG. 19 Panel A, lane 2). The fact that the apparentmolecular weight of p49 is greater than the expected value of ˜43-kDamay be attributable to glycosylation. In contrast, a 20-kD protein wassecreted when PDGF D-transfected cells were grown in the presence of FBS(FIG. 19 Panel A, lane 3). Conditioned media from mock transfected cellsdid not react with the anti-V5 antibody (FIG. 19 Panel A, lane 1).

In addition, PDGF D was expressed in the presence or absence of FBS andpurified to >95% homogeneity. As shown in FIG. 19 Panel B (lane 2),expression of PDGF D under serum-free conditions resulted in thedetection of the expected 49-kD gene product under reducing conditions,when the gel was stained using Coomassie Blue. A polypeptide specieswith an apparent molecular weight of about 84 kDa, corresponding to adimeric p85 species of p49, was seen under non-reducing conditions (FIG.19 Panel B, lane 1). When PDGF DD was purified from serum-containingconditioned medium and run under nonreducing conditions, a species withan apparent molecular weight of about 35 kDa (p35) was observed (FIG. 19Panel B, lane 3). Under reducing conditions, p35 was found to yieldthree bands when visualized with Coomassie Blue, which migrate withapparent molecular weights of approximately 20, 14, and 6 kDa (FIG. 19Panel B, lane 4).

Amino terminal sequence analysis of p35 demonstrated proteolyticcleavage after Arg247 (R247) or Arg249 (R249) (FIG. 20). As indicated inPanel A of FIG. 20, two peptides were found, one beginning with GlyArg(GRSYHDR . . . ; shown with the GR residues underlined), and the secondbeginning with the third residue, Ser (SYHDR . . . ). The ratio of thesepeptides was found to be SYHDR:GRSYHDR=4:1. The additional sequencingresults in FIG. 20 (Panels B and C) indicate that further processingproduces the remaining polypeptides seen with Coomassie blue stainingbut not with anti-V5 Westerns, namely the 16 kDa and 6 kDa speciesshown. These are joined together to provide p35.

The results presented in this Example indicate that the PDGF D geneproducts are dimers in both the holoprotein form (p85) and theC-terminal fragment (p35). The p85 form appears to be processed in thepresence of FBS to provide the p35 form. These dimeric forms aredesignated PDGF DD.

Example 22 Processing of the 30664188 Gene Product in the Presence ofFetal Bovine Serum and Calf Serum

The 30664188 gene product was incubated in the presence of increasingconcentrations of calf serum (FIG. 21, Panel A) or fetal bovine serum(Panel B). The results demonstrate that only fetal bovine serum (PanelB) but not calf serum (Panel A) processes the p85 form of the 30664188gene product to provide p35.

Example 23 Stimulation of Growth of Pulmonary Artery Smooth Muscle Cellsby Growth Factors

This Example demonstrates the ability of PDGF DD to stimulate growth ofpulmonary artery smooth muscle cells.

The p35 dimer of 30664188, PDGF AA or PDGF BB were added at variousconcentrations to pulmonary artery smooth muscle cells (Clonetics) afterbeing cultured in 6-well plates to approximately 35% confluence, washedwith DMEM, and starved overnight. After 18 hrs, BrdU was added, and 5hrs later the cells were analyzed for BrdU incorporation using aBrdU-directed ELISA.

The results are shown in FIG. 22. It is seen that the maximal effectachieved by treatment with p35 dimer exceeds that given by both PDGF AAand PDGF BB. It is seen that the effects of p35 dimer and PDGF BBresemble each other more closely than the effect obtained with PDGF AA.Of all three growth factors tested, p35 dimer induced the greatestgrowth in smooth muscle cells, as determined by BrdU incorporation, with50% maximal effect obtained at less than 12.5 ng/mL.

Example 24 Proliferation of Pulmonary Artery Smooth Muscle Cells inResponse to Various Growth-Promoting Treatments

This Example demonstrates the ability of PDGF DD to stimulateproliferation of pulmonary artery smooth muscle cells.

Pulmonary artery smooth muscle cells were cultured in 6-well plates toapproximately 35% confluence, washed with DMEM, and starved overnight.Cells were then fed with DMEM supplemented with recombinant 30664188, aknown PDGF (200 ng/ml) or 10% FBS for three days. Culture fluids wereremoved and replaced with same media for an additional 2-3 days. Toquantitate the smooth muscle cell growth assay, cells were trypsinizedand counted with a Beckman Coulter Z1 series counter (Beckman Coulter,Fullerton, Calif.).

The results are shown in FIG. 23. It is seen that PDGF produces a modestincrease in cell number, whereas treatment with 30664188 provides aneffect, compared with control, that is almost double that observed withPDGF. A positive control using treatment with 10% FBS gave a verypronounced effect. Treatment of smooth muscle cells with 30664188 andPDGF BB led to elongated bipolar spindle shaped phenotype in contrast tothe flat club shaped phenotype observed with serum.

30664188 is an effective stimulant of pulmonary artery smooth musclecell proliferation, and suggests that 30664188 has a therapeutic use inwound healing, tissue repair and cartilage repair. Furthermore,antibodies directed against 30664188 may have therapeutic use ininhibiting or preventing restenosis of patent vasculature.

Example 25 Proliferation of Saphenous Vein Cells in Response to VariousGrowth-Promoting Treatments

This Example illustrates the ability of PDGF DD to stimulateproliferation in saphenous vein cells. Saphenous vein cells (Clonetics)were treated and analyzed as described in Example 24. The results (notshown) indicate that PDGF produces a slightly lower increase in cellnumber than does treatment with 30664188, which provides proliferationto almost 5 times the cell number seen with the control. A positivecontrol using treatment with 10% FBS gave a very pronounced effect.30664188 is an effective stimulant of saphenous vein cell proliferation,and suggests that 30664188 and 30664188 antibodies has a therapeutic usein wound healing, tissue repair and cartilage repair. Furthermore,antibodies directed against 30664188 may have therapeutic use ininhibiting or preventing restenosis of patent vasculature.

Example 26 Mouse Model for Inflammatory Bowel Disease

A widely recognized animal model for inflammatory bowel disease is themouse dosed with the sodium form of dextran sulfate.

Materials and Methods

Colitis Study Design. Normal female Balb/c mice (Harlan Labs), 6-8 weeksold weighing approximately 20 g, were housed 3-5 animals per cage inpolycarbonate cages with filter tops and given food (Harlan Teklad mousechow) and tap water ad libitum. Mice were acclimated for 6 days (Day-7through Day-1) and then given water orally (po) ad libitum containing 5%dextran sulfate sodium (DSS) or control water ad libitum for 7 days (Day0 through Day 6). DSS (Spectrum Chemicals, Gardena Calif.) was made as a5% solution in tap water; DSS was made every other day and stored at 4°C. Mice were divided into 4 treatment groups (Table 14). On Day 0, dailyintraperitoneal (ip) treatments with vehicle (1M L-arginine in phosphatebuffered saline) or protein (CG53135 or CG52053, 5 mg/kg) were initiatedand continued each morning through Day 6. On Day 7, mice were sacrificedby exposure to CO₂.

TABLE 14 Treatment Groups Group N^(a) Treatment 1 5 Normal control: noDSS water + vehicle ip 2 10 Disease control: DSS water po + vehicle ip 310 CG53135: DSS water po + 5 mg/kg CG 53135 ip 4 5 DSS water po + 5mg/kgCG52053 ip ^(a)N = number of animals per group

Protein production. The cDNA for CG52053 was identified and cloned intothe pCEP4/Sec vector (Invitrogen, Carlsbad, Calif.) and transfected intohuman embryonic kidney cells (HEK 293). The transfected cells wereselected using Hygromycin B and then scaled up in 10 L bioreactors usingDMEM medium containing 10% FBS. The CG52053 protein was purified fromthe culture medium by ion-exchange and metal affinity chromatography.The final purified CG52053 was diluted in 20 mM Tris HCl (pH 7.4) and 50mM NaCl.

The cDNA for CG53135 was identified and cloned into the pRSET vector(Invitrogen) to provide the vector pETMY-FGF-CX described in Example 5.The gene product of this construct provides a polypeptide incorporating(His)₆-(enterokinase cleavage site)-(multicloning site) at theN-terminal end of the polypeptide; in addition, in this construct, theFGF-CX sequence begins with the Ala at position 2 of Table 1 (SEQ IDNO:2). This vector was transformed into Escherichia coli. The E. colicells were grown up to 10 L scale and infected with CE6 phage to producethe recombinant CG53135. The recombinant protein was purified bydisrupting the E. coli cells in a microfluidizer and extraction with 1ML-arginine solution, followed by multiple metal affinity chromatographysteps. The final purified protein was dialyzed into phosphate bufferedsaline containing 1M L-arginine. Protein purity was determined bySDS-PAGE analysis and identities were confirmed by Western blotanalysis. Activity of proteins was determined by BrdU incorporationassay (Roche Molecular Biochemicals) using a 5 hr incorporation time andNIH 3T3 cells.

Body weights were measured daily and at termination on day 7. Additionalparameters measured at necropsy included colon length, colon weight andspleen weights. Colon and spleen were collected into formalin forhistopathologic evaluation.

Colon content was scored at necropsy according to the followingcriteria:

0=normal to semi-solid stool, no blood observed

1=normal to semi-solid stool, blood tinged

2=semi-solid to fluid stool with definite evidence of blood

3=bloody fluid

Pathology Methods

Three sections approximately 1 cm apart from the distal end (area thatis most severely affected in this model) and 3 sections approximately 1cm apart from the proximal end (less severely affected area) wereprocessed for paraffin embedding, sectioned and stained with hematoxylinand eosin for pathologic evaluation.

For each section, submucosal edema was quantitated by measuring thedistance from the muscularis mucosa to the internal border of the outermuscle layer. Inflammation (foamy macrophage, lymphocyte and PMNinfiltrate) was assigned severity scores according to the following:

Normal=0

Minimal=1

Mild=2

Moderate=3

Marked=4

Severe=5

Splenic lymphoid atrophy was also scored by the above criteria.

The parameters reflecting epithelial cell loss/damage were scoredindividually using a % area involved scoring method:

None=0

1-10% of the mucosa affected=1

11-25% of the mucosa affected=2

26-50% of the mucosa affected=3

51-75% of the mucosa affected=4

76-100% of the mucosa affected=5

Parameters that were scored using % involvement included:

Colon glandular epithelial loss—this includes crypt epithelial as wellas remaining gland epithelial loss and would equate to crypt damagescore.

Colon Erosion—this reflects loss of surface epithelium and generally wasassociated with mucosal hemorrhage (reflective of the bleeding seenclinically and at necropsy).

For each animal, 3 proximal (less severe lesions) and 3 distal (mostsevere lesion) areas were scored and the mean of the scores for each ofthe regions was determined. Group means and % inhibition from diseasecontrol were determined. By doing it this way (rather than summing thescores from various sections) one can look at the mean±SE for inindividual Aparameter (represented by 3 sections) and equate it to adelineated severity. As an example, if the mean is 4 for glandepithelial loss one knows that 51-75% of the mucosa was devoid ofepithelium.

The 3 important scored parameters (inflammation, glandular epithelialloss, erosion) were ultimately summed to arrive at a sum ofhistopathology score which indicates the overall damage and would have amaximum score of 15.

One final summation of proximal+distal summed scores was done to reflectthe overall total colonic severity score.

Statistics. The mean and standard error (SE) for each treatment groupwas determined for each parameter scored; the data were compared to thedata for the disease controls (Group 2) using a 2-tailed Student's ttest with significance at p≦0.05.

Results

Live Phase, Necropsy and Organ Weight

All animals except DSS+vehicle control mouse 4 survived to studytermination. Mouse 4 was found dead the morning of necropsy on day 7.

DSS treatment-related changes in body weight were present by day 3 inall DSS treated mice. At study termination, DSS+vehicle controls had a25% decrease in body weight (FIGS. 24, 25 and 26). A significantbeneficial effect on DSS induced weight loss was seen in mice givenFGF-CX, referred to as AB020858 (FIGS. 25 and 26).

Clinical evidence of bloody diarrhea was evident in all DSS+vehicleanimals except animal 1. At necropsy all DSS controls had blood or bloodtinged fluid in the colon. In contrast, mice treated with AB020858generally had semi-solid stool and little evidence of blood. Similarfindings occurred in mice treated with 30664188.

Colon content scores reflecting colonic hemorrhage were dramaticallydecreased (93%) in mice treated with AB020858 and (79%) in mice treatedwith 30664188 (FIG. 34).

Absolute spleen weights (FIGS. 27 and 28) were decreased approximately30% in mice treated with vehicle. Treatment with AB020858 resulted in55% reduction of the DSS-induced losses in spleen weights. Treatmentwith 30664188 reduced the splenic weight losses by 62%.

Absolute colon weights (FIGS. 29 and 30) were decreased approximately26% in mice treated with vehicle. Treatment with AB020858 resulted inslight but not significant reduction of the DSS-induced changes in colonweights. Treatment with 30664188 reversed the colon weight decreases(FIGS. 30 and 31).

Absolute colon lengths (FIGS. 32 and 33) were decreased approximately40% in mice treated with DSS+vehicle. Treatment with AB020858 resultedin significant (40%) reduction of the DSS-induced changes in colonlength. Treatment with 30664188 reduced the colon length loss 36%.

Histopathology Findings

Histopathology was conducted on the full length of the colon. Lesionswere much greater in the distal vs. proximal colon, as expected.Quantitation of efficacy of treatment is based primarily on inhibitionof pathological changes in this location. Colonic edema in the distalcolon was inhibited 76% by treatment with AB020858 whereas treatmentwith 30664188 did not inhibit the edema (FIG. 35).

Colonic inflammation in the distal colon was inhibited 55% by treatmentwith AB020858 and 41% by treatment with 30664188 (FIG. 36).

Protection of colonic epithelium (both crypts and remainder of thegland), as determined by the epithelial loss score, was 57% in micegiven AB020858 and 41% in those treated with 30664188 (FIG. 37). Furtherevidence of mucosal epithelial protection in the distal colon wasevident on evaluation of degree of surface epithelial loss leading toerosion/ulceration. As shown by the colon erosion scores, AB020858treatment gave 84% inhibition of the erosive lesions and 30664188treatment resulted in 74% inhibition (FIG. 38).

Summing the important histologic scores for inflammation, glandularepithelial damage and erosion (FIG. 39), it is seen that an overallprotective effect results from the treatment with AB020858, whichprovides 66% inhibition of the pathology. Treatment with 30664188resulted in 53% inhibition of the overall score. Slight but notsignificant (33-37%) inhibition of the total histologic scores wasevident for proximal colon. Results for the colon overall are shown inFIG. 40.

Splenic weight decreases were largely a result of splenic lymphoidatrophy. Treatment with both proteins inhibited this parameter as well(FIG. 41).

Discussion and Conclusions

In this model of inflammatory bowel disease, in which mice are exposedto 5% DSS for 7 days, most animals develop marked to severe distalcolonic inflammation/edema in association with crypt and colonicglandular epithelial loss and erosion/ulceration leading to markedhemorrhage. Lesions in the proximal colon are much milder but similar incharacter.

Cotemporaneous treatment with AB020858 (5 mg/kg, qd, d0-6) resulted inclinical benefit (reduced body weight loss) as well as protectionagainst development of hemorrhagic diarrhea, a common feature of thismodel. Stressed unhealthy DSS treated mice have splenic lymphoidatrophy. This parameter (reflected by weight changes and histologicalterations) was also benefited by treatment with AB020858.

Colonic shortening (due to inflammation and mucosal tissue loss) wasinhibited 40% by treatment with AB020858. This gross observation wasstrongly supported by the histologic observations of mucosal epithelialpreservation in the crypts, colonic glands and surface epithelium (seeFIGS. 42 and 43). In FIG. 42, viewed at 400× in the original images, thenormal colonic mucosa has uniform glandular architecture and nosubmucosal edema (upper left). The disease control has no mucosal glandsand surface epithelium, exposing blood vessels of the severely inflamedlamina propria to the lumen and resulting in hemorrhage (upper right).treatment with CG53135 preserves mucosal integrity and results indecreased epithelial loss and reduced inflammation in the lamina propria(lower left). Treatment with CG52053 decreases epithelial loss andmucosal inflammation, although to a lesser degree than treatment withCG53135 (lower right). In FIG. 43, viewed at 50× in the original images,the normal control shows normal colonic mucosa with uniform glandulararchitecture and no submucosal edema (upper left). DSS-induced colitisresults in loss of glandular architecture and edema that separates themucosa from the outer muscle layers (upper right). Treatment withCG53135 inhibits the severe mucosal changes and submucosal edema inducedby DSS (lower left). Treatment with CG52053 results in some inhibitionof inflammation and loss of glandular architecture but no inhibition ofsubmucosal edema (lower right). This histologic evidence of mucosalprotection corroborates the dramatic necropsy observation that verylittle hemorrhagic diarrhea occurs.

The results of the experiments reported in this Example indicate that,in mice in which inflammatory bowel disease is induced by oraladministration of DSS for 7 days, simultaneous treatment with the growthfactors employed here during the course of exposure to DSS led tosignificant therapeutic benefits compared to untreated DSS controls.

Example 27 Dose Responsive Effects of AB020858 Female Swiss Webster Micewith Dextran Sulfate-induced Colitis

The experiments reported in this Example report the results of dosetitration experiments in an animal model of inflammatory bowel diseaseusing a different strain of mouse than that used in Example 26.

Introduction and General Methods

Colitis Study Design. Normal female Swiss-Webster mice (Harlan Labs),6-8 weeks old weighing approximately 20 g, were acclimated for 4 days(Day-4 through Day-1) and then given water orally (po) ad libitumcontaining 5% dextran sulfate sodium (DSS) or control water ad libitumfor 7 days (Day 0 through Day 6). DSS (Spectrum Chemicals, GardenaCalif.) was made as a 5% solution in tap water; DSS was made every otherday and stored at 4° C. Mice were divided into 8 treatment groupsincluding QD doses of 0.3, 1, 3 and 10 mg/kg, and a BID dose regimen of5 mg/kg per dose (Table 15). On Day 0, daily intraperitoneal (ip)treatments with vehicle (1M L-arginine in phosphate buffered saline) orCG53135 protein in vehicle were initiated and continued through Day 6.On Day 7, mice were sacrificed with CO₂.

TABLE 15 Treatment Groups Disease Disease Treatment Normal Control^(b)CG53135 CG53135 CG53135 CG53135 Control^(b) CG53135 Group Control^(a) QDQD QD QD QD BID BID Group # 1 2 3 4 5 6 7 8 CG 53135 0 0 10 3 1 0.3 0 5(mg/kg) Number of 4 10 10 10 10 10 10 10 Test Animals ^(a)normal control= vehicle only; ^(b)disease control = 5% DSS + vehicle

Protein production. The CG53135 protein was produced in E. coli asdescribed in Example 26. The recombinant protein was purified bydisrupting the E. coli cells (resuspended in a 1 M L-arginine solution)in a microfluidizer, followed by multiple metal affinity chromatographysteps. The final purified protein was dialyzed into phosphate bufferedsaline containing 1M L-arginine.

Colon content was scored as described in Example 1.

Pathology Methods

Three sections equidistant apart from the distal one third of the colon(area that is most severely affected in this model) were processed forparaffin embedding, sectioned and stained with hematoxylin and eosin forpathologic evaluation.

For each section, scoring was done as described in Example 26.

Splenic lymphoid atrophy was also scored by the above criteria.

Epithelial cell loss/damage was scored as described in Example 26.

Parameters that were scored using % involvement included:

Colon glandular epithelial loss—this includes crypt epithelial as wellas remaining gland epithelial loss and would equate to crypt damagescore.

Colon Erosion—this reflects loss of surface epithelium and generally wasassociated with mucosal hemorrhage (reflective of the bleeding seenclinically and at necropsy).

For each animal, 3 distal (most severe lesion) areas were scored.Scoring and analysis was done as described in Example 26.

Live Phase, Necropsy and Organ Weight Results

Four animals died during the course of the study (#10 in vehicle controlgroup 2 on day 7, #3 in group 6, 0.3 mg/kg on day 6, #5 in group 8vehicle control BID on day 7, and #6 in group 7 5 mg/kg BID on day 6).

DSS treatment-related changes in body weight were obvious by day 5 inall DSS treated mice and ultimately were most severe in animals treatedwith vehicle (FIG. 44). At study termination, DSS+vehicle controls had a28% decrease in body weight. A significant beneficial effect on DSSinduced weight loss was seen in mice given AB020858 QD at all doses(FIG. 45).

Clinical evidence of bloody diarrhea was evident in all DSS+vehicleanimals. At necropsy all DSS controls had blood or blood tinged fluid inthe colon. In contrast, mice treated QD with 10 mg/kg AB020858 generallyhad semi-solid stool and less blood (except animals #5). Clinicalbenefit was also evident but less impressive in those given doses of 3or 1 mg/kg QD and absent in those treated with 0.3 mg/kg (FIG. 46). Micetreated BID with 5 mg/kg had the most impressive clinical benefit (68%inhibition) and clinically these mice had the best overall improvement.

Absolute colon lengths (FIGS. 47 and 48) were decreased 41% in micetreated with vehicle. Treatment with AB020858 QD at 10 mg/kg resulted insignificant (21%) inhibition of the DSS-induced changes in colon length.Treatment with AB020858 BID at 5 mg/kg reduced the colon length decrease36%.

Absolute colon weights (FIGS. 49 and 50) were decreased approximately26% in mice treated with DSS in vehicle. Treatment with AB020858 at 10mg/kg QD or 5 mg/kg BID resulted in significant reduction of theDSS-induced changes in colon weights.

Absolute spleen weights (FIG. 51) were increased approximately 40% inmice treated with DSS+vehicle (due to extreme extramedullaryhematopoiesis). Spleen weights were significantly greater in all DSStreated animals vs. normal.

Histopathology Findings

Significant reduction of colonic inflammation, gland loss, erosion andtotal histopathology scores occurred in mice treated with AB020858 QD(10 mg/kg) and BID (5 mg/kg) and was of approximately equal magnitude(FIGS. 52, 53, 54 and 55).

Splenic lymphoid atrophy (an indication of stress) was inhibited inthese same animals 47% and 46% respectively (FIG. 56). Inhibition ofinduction of splenic extramedullary hematopoiesis was greater in micetreated BID vs. QD and occurred in all treatment groups (FIG. 57).

Discussion and Conclusions

The experiments reported in this Example provide dose-responseinformation for the administration of AB020858, using a different strainof mouse than those in Example 26 (which used Balb/c mice). The resultsindicate that simultaneous administration of AB020858 is effective ininhibiting the appearance of markers of DSS-induced inflammatory boweldisease, especially with the highest doses used.

Example 28 Administering CG53135 Subcutaneously

An additional experiment was carried out in which mice were also treatedsubcutaneously with CG531135. Together with the results in Examples 26and 27, these studies demonstrate that prophylactic administration ofCG53135 at doses of 5 or 10 mg/kg ip and 5 or 1 mg/kg sc significantlyreduce the extent and severity of mucosal damage induced by dextransulfate sodium in a murine model of colitis.

Example 29 Effects of Administering CG53135 to Indomethacin-treated Rats

Treatment of rats with indomethacin results in gross and histopathologicintestinal alterations that are similar to those occurring in Crohn'sDisease. The experiments provided in this Example report on the efficacyof CG53135 in treating the rat model of indomethacin-induced intestinalinjury. The efficacy of this protein in an alternate model of intestinalinjury adds support to the therapeutic potential of CG53135 in treatmentof inflammatory bowel disease.

Materials & Methods

Protein production. Preparation of CG53135 protein was the same asdescribed in Example 26.

Study Design. Female Lewis rats (Harlan, Indianapolis, Ind.) weighing175-200 g were acclimated for 8 days (Day-8 through Day-1). Rats weredivided into 8 treatment groups: four groups receiving CG53135 (threegroups iv and one group sc), two iv controls for normal and the diseasemodel, and two sc controls for normal and the disease model. On Day-1,treatments with CG53135 or vehicle were initiated and continued throughDay 4. CG53135 was injected iv (tail vein) at doses of 5, 1 or 0.2mg/kg, or 1 mg/kg sc; vehicle controls were injected with BSA (5 mg/mLin PBS+1M L-arginine). On Days 0 and 1 rats were treated withindomethacin (Sigma Chemical Co., St. Louis, Mo.; 7.5 mg/kg doses) inorder to induce gross and histopathologic intestinal alterations similarto those occurring in Crohn's Disease. Indomethacin was prepared in 5%sodium bicarbonate. On Day 5, rats were injected with a single ip doseof 50 mg/kg 5-bromo-2′deoxyuridine (BrdU, Calbiochem, LaJolla, Calif.) 1hour prior to necropsy in order to pulse label proliferating cells inthe intestine and spleen. Following termination, a 10 cm section ofjejunum in the area at risk for lesions was weighed, given a grosspathology score, and then collected into formalin for histopathologicevaluation and scoring of necrosis and inflammation. Blood was collectedfor CBC analysis.

Observations and Analysis of Markers of Pathology

Gross Observations. Body weight was measured daily beginning on Day 0.At necropsy, liver and spleen weights were measured, and a 10 cm sectionof jejunum in the area at risk was weighed, scored for gross pathology,and collected into formalin for histopathologic evaluation and scoringof necrosis and inflammation. The area at risk for indomethacin-inducedinjury was scored at necropsy according to the following criteria:

0=normal

1=minimal thickening of the mesentery/mesenteric border of the intestine

2=mild to moderate thickening of the mesentery/mesenteric border ofintestine, but no adhesions

3=moderate thickening with 1 or more definite adhesions that are easilyseparated

4=marked thickening with 1 to numerous hard to separate adhesions

5=severe intestinal lesions resulting in death.

Histopathology. Five sections (approximately equally spaced) taken fromthe weighed 10 cm area at risk of small intestine forindomethacin-induced lesions were fixed in 10% neutral bufferedformalin, processed for paraffin embedding, sectioned at 5 μm andstained with hematoxylin and eosin for histopathologic evaluation.Necrosis was scored according to the percent area of the sectionaffected in the same way as described in Example 26 for scoringepithelial cell loss.

Inflammation was scored according to the following criteria:

0=none

1=minimal inflammation in mesentery and muscle or lesion

2=mild inflammation in mesentery and muscle or lesion

3=moderate inflammation in mesentery and muscle or lesion

4=marked inflammation in lesion

5=severe inflammation in lesion.

The means for inflammation and necrosis were determined for each animal,and then the means for each group were calculated.

Statistics. The mean and standard error (SE) for each treatment groupwere determined for each parameter scored; the data were compared to thedata for the disease controls using a 2-tailed Student's T test withsignificance at p<0.05.

Results

Weight loss was observed in all animals treated with indomethacin. Aslight, but significant reduction in weight loss was observed in animalstreated with CG53135 (0.2 mg/kg iv) as compared with disease controls(iv). Other doses of CG53135 (both iv and sc routes of administration)provided diminished, but not statistically significant,indomethacin-induced weight loss (FIG. 58).

At necropsy, a 10 cm section of jejunum in the area at risk from eachanimal was weighed. Indomethacin treatment resulted in an elevation insmall intestine weight as compared with normal iv and sc controls,consistent with edema and inflammation associated with this model ofintestinal injury. Treatment with CG53135 (1 mg/kg or 0.2 mg/kg iv)resulted in significant reductions in small intestine weight as comparedwith disease controls (FIG. 59). A slight reduction in the smallintestine clinical score was observed, with the greatest benefitoccurring with the 1.0 mg/kg iv dose (38%) and the 0.2 mg/kg iv dose(25%); these benefits, however, were not statistically significant.Relative spleen and liver weights were increased in animals treated withindomethacin. Administration of CG53135 produced moderate additionalincreases in these weights (data not shown).

Hematology. Administration of 2 doses of indomethacin to rats increasedthe total white blood cell count as a result of increased neutrophilsand lymphocytes. Reductions in red blood cell count, hematocrit, andhemoglobin concentration were also observed. Treatment with CG53135 (5mg/kg and 0.2 mg/kg iv) resulted in significant reductions in absoluteneutrophil counts as compared with disease controls (FIG. 60).Hemoglobin concentration was diminished in the indomethacin controlscompared to normal controls, and slightly further diminished in ratestreated with CG53135 (data not shown).

Histopathology. Evaluation and scoring of 5 sections of intestine wereconducted for each animal. Histologic evidence of a protective effect onthe intestine was observed only in animals treated with CG53135 (0.2mg/kg iv). A 53% reduction in jejunal necrosis and 38% reduction ininflammation score were observed for the 0.2 mg/kg iv CG53135 dose ascompared with disease controls iv (FIG. 61). Photomicrographs ofaffected small intestine are shown in FIG. 62 for a normal and diseasecontrol, and a rat treated with 0.2 mg/kg CG53135. Panel A shows thesmall intestine from a normal control animal treated iv with vehicle(BSA). Normal villous architecture and mesentery (arrow) are apparent.Panel B presents a photomicrograph of the small intestine from anindomethacin-treated rat, with vehicle (BSA) iv. Focal mucosal necrosisextending across most of the area associated with attachment of themesentery is apparent (see, for example, the asterisks at upper rightintestinal wall and lower right intestinal wall). Marked inflammatorycell infiltrate is present in the mesentery (arrow). Panel C shows theimage of the small intestine from an indomethacin-treated rat furthertreated with CG53135, 0.2 mg/kg iv. There is no apparent necrosis, incontrast to the disease control shown in Panel B. There is a focal areaof attenuated villi and cellular infiltration into muscle layer (see,for example, the three asterisks at the upper right, right and lowerright of the intestinal wall). Mesentery (arrow) is infiltrated byinflammatory cells. The photomicrographs in FIG. 62 provide furthersupport for the protective effect of 0.2 mg/kg iv CG53135

BrdU labeling was carried out by injecting 50 mg/kg 1 hr prior tonecropsy. In the small intestine from a normal control animal, normalpattern of crypt labeling is seen at 100× (FIG. 63, Panel A). BrdUincorporation in the disease model was decreased or absent ineptithelial cells in mucosal areas of necrosis, but increased insubajacent inflammatory tissue in which fibroblast labeling wasprominent (FIG. 63, Panel B, visualized at 50×). Focal mucosal necrosis(arrow) is delineated by an absence of BrdU immunostaining as well assevere infiltration of inflammatory cells and fibroblast proliferation.Small intestine from a rat treated with indomethacin+CG53135 0.2 mg/kgiv shows an absence of crypt labeling, but relatively intact mucosa(arrow in FIG. 63, Panel C, visualized at 50×). Subadjacent smoothmuscle and mesentery is only mildly infiltrated with inflammatory cells,compared with that seen in the disease control (Panel B). In certainanimals treated with CG53135, in which preservation of mucosal integrityoccurred, increased crypt labeling was also observed; this is in thedirection found in the normal control.

The results of the experiments in this Example may be summarized asfollows. Treatment of rats with indomethacin results in gross andhistopathologic intestinal alterations that are similar to thoseoccurring in Crohn's Disease. Administration of CG53135 (0.2 mg/kg iv)to indomethacin-treated rats resulted in significant reductions inweight loss, small intestine weight, absolute neutrophil counts, andjejunal necrosis and inflammation scores. Higher doses of CG53135 (5, 1mg/kg iv and 1 mg/kg sc) were less efficacious. The morphologicalappearance of tissues collected from animals injected with BrdU 1 hourprior to necropsy suggested that the beneficial effects of CG53135 inthis model of intestinal injury were the result of mucosal protectionrather than a proliferative effect on target cells.

Example 30 Therapeutic Administration of CG53135 Enhances Survival inthe Murine DSS Model

In the experiments described in Examples 26-28, DSS exposure and CG53135administration were initiated simultaneously on day 0. In the presentExample, the effect of CG53135 administered after the initiation of DSStreatment was examined. CG53135 was prepared as described in Example 26.Balb/c mice were exposed to DSS for 7 days (day 0 to day 6). The micewere injected daily subcutaneously with various concentrations ofCG53135 (5, 1 and 0.2 mg/kg) beginning on the fifth day of DSS exposure(i.e. day 4) and ending 3 days after the termination of DSS exposure(i.e. day 9), or with vehicle only. Animal survival was recorded on adaily basis and the experiment was concluded on day 10. As shown in FIG.64, therapeutic administration of CG53135 at 5 mg/kg enhanced survivalrelative to the disease control group. Thus, while only 44% (4 of 9) ofthe animals in the disease control group survived until the end of thestudy, 89% (8 of 9) of the animals treated with CG53135 at 5 mg/kgsurvived. CG53135 administered therapeutically at lower doses had noeffect on survival relative to control.

EQUIVALENTS

From the foregoing detailed description of the specific embodiments ofthe invention, it should be apparent that particular novel compositionsand methods involving nucleic acids, polypeptides, antibodies, detectionand treatment have been described. Although these particular embodimentshave been disclosed herein in detail, this has been done by way ofexample for purposes of illustration only, and is not intended to belimiting with respect to the scope of the appended claims that follow.In particular, it is contemplated by the inventors that varioussubstitutions, alterations, and modifications may be made as a matter ofroutine for a person of ordinary skill in the art to the inventionwithout departing from the spirit and scope of the invention as definedby the claims. Indeed, various modifications of the invention inaddition to those described herein will become apparent to those skilledin the art from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. A method of ameliorating a symptom associated with an inflammatorybowel disease comprising administering to a subject an amount effectiveto ameliorate said inflammatory disease of a polypeptide comprising anamino acid SEQ ID NO:
 2. 2. A method of ameliorating a symptomassociated with an inflammatory bowel disease comprising administeringto a subject a compsition comprising a pharmaceutically acceptablecarrier and an amount effective to ameliorate said inflammatory diseaseof a polypeptide comprising an amino acid SEQ ID NO:
 2. 3. A method oftreating an inflammatory bowel disease comprising administering to asubject an amount effective to treat said inflammatory bowel disease ofa polypeptide comprising an amino acid sequence of SEQ ID NO:2.
 4. Amethod of treating an inflammatory bowel disease comprisingadministering to a subject a composition comprising a pharmaceuticallyacceptable carrier and an amount effective to treat said inflammatorybowel disease of a polypeptide comprising an amino acid sequence of SEQID NO:2.
 5. The method of claim 3 or 4, wherein said inflammatory boweldisease is Crohn's disease.
 6. The method of claim 3 or 4, wherein saidinflammatory bowel disease is ulcerative colitis.
 7. The method of claim3 or 4, wherein said administering is parenteral administration.
 8. Themethod of claim 7, wherein said parenteral administration is intravenousadministration.
 9. The method of claim 7, wherein said parenteraladministration is subcutaneous administration.
 10. The method of claim 3or 4, wherein said administration is rectal administration.
 11. Themethod of claim 3 or 4, wherein said administration is transdermaladministration.
 12. The method of claim 3 or 4, wherein saidadministration is transmucosal administration.
 13. The method of claim12, wherein said transmucosal administration is nasal administration.14. The method of claim 3 or 4, wherein said subject is a mammal. 15.The method of claim 3 or 4, wherein said subject is a human.
 16. Themethod of claim 1 or 2, wherein said inflammatory bowel disease isCrohn's disease.
 17. The method of claim 1 or 2, wherein saidinflammatory bowel disease is ulcerative colitis.
 18. The method ofclaim 1 or 2, wherein said administering is parenteral administration.19. The method of claim 18, wherein said parenteral administration isintravenous administration.
 20. The method of claim 18, wherein saidparenteral administration is subcutaneous administration.
 21. The methodof claim 1 or 2, wherein said administration is rectal administration.22. The method of claim 1 or 2, wherein said administration istransdermal administration.
 23. The method of claim 1 or 2, wherein saidadministration is transmucosal adm`inistration.
 24. The method of claim23, wherein said transmucosal administration is nasal administration.25. The method of claim 1 or 2, wherein said subject is a mammal. 26.The method of claim 1 or 2, wherein said subject is a human.